Stabilized group 2 influenza hemagglutinin stem region trimers and uses thereof

ABSTRACT

Vaccines that elicit broadly protective anti-influenza antibodies. The vaccines comprise nanoparticles that display HA trimers from Group 2 influenza virus on their surface. The nanoparticles are fusion proteins comprising a monomeric subunit (e.g., ferritin) joined to stabilized stem regions of Group 2 influenza virus HA proteins. The fusion proteins self-assemble to form the HA-displaying nanoparticles. Also provided are fusion proteins, and nucleic acid molecules encoding such proteins, and assays using nanoparticles of the invention to detect anti-influenza antibodies.

CROSS REFERENCE TO RELATED APPLICATION

This application is a national stage application under 35 U.S.C. 371 ofPCT Application No. PCT/US2017/049894 having an international filingdate of Sep. 1, 2017, which designated the United States, which PCTapplication claimed the benefit of U.S. Provisional Patent ApplicationSer. No. 62/383,267 filed on Sep. 2, 2016, the disclosures of each ofwhich are incorporated herein by reference in their entireties.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted as an electronictext file named “Sequence_Listing_ST25.txt”, having a size in bytes of452 KB, and created on May 18, 2021. The information contained in thiselectronic file is hereby incorporated by reference in its entiretypursuant to 37 CFR § 1.52(e)(5).

BACKGROUND

Protective immune responses induced by vaccination against influenzaviruses are primarily directed to the viral HA protein, which is aglycoprotein on the surface of the virus responsible for interaction ofthe virus with host cell receptors. HA proteins on the virus surface aretrimers of HA protein monomers that are enzymatically cleaved to yieldamino-terminal HA1 and carboxyl-terminal HA2 polypeptides. The globularhead consists exclusively of the major portion of the HA1 polypeptide,whereas the stem that anchors the HA protein into the viral lipidenvelope is comprised of HA2 and part of HA1. The globular head of a HAprotein includes two domains: the receptor binding domain (RBD), an˜148-amino acid residue domain that includes the sialic acid-bindingsite, and the vestigial esterase domain, a smaller ˜75-amino acidresidue region just below the RBD. The globular head includes severalantigenic sites that include immunodominant epitopes. Examples includethe Sa, Sb, Ca₁, Ca₂ and Cb antigenic sites (see, for example, Caton, etal, 1982, Cell 31, 417-427). The RBD-A region includes the Sa antigenicsite and part of the Sb antigenic site.

Antibodies against influenza often target variable antigenic sites inthe globular head of HA, which surround a conserved sialic acid bindingsite, and thus, neutralize only antigenically closely related viruses.The variability of the HA head is due to the constant antigenic drift ofinfluenza viruses and is responsible for seasonal endemics of influenza.In contrast, the HA stem is highly conserved and experiences littleantigenic drift. Unfortunately, unlike the immunodominant head, theconserved HA stem is not very immunogenic. Furthermore, gene segments ofthe viral genome can undergo reassortment (antigenic shift) in hostspecies, creating new viruses with altered antigenicity that are capableof becoming pandemics [Salomon, R. et al. Cell 136, 402-410 (2009)].Until now, each year, influenza vaccine is updated to reflect thepredicted HA and neuraminidase (NA) for upcoming circulating viruses.

Recently, an entirely new class of broadly neutralizing antibodiesagainst influenza viruses was isolated that recognize the highlyconserved HA stem [Corti, D. et al. J Clin Invest 120, 1663-1673 (2010);Ekiert, D. C. et al. Science 324, 246-251 (2009); Kashyap, A. K. et al.Proc Natl Acad Sci USA 105, 5986-5991 (2008); Okuno, Y. et al. J Virol67, 2552-2558 (1993); Sui, J. et al. Nat Struct Mol Biol 16, 265-273(2009); Ekiert, D. C. et al. Science 333, 843-850 (2011); Corti, D. etal. Science 333, 850-856 (2011)]. Unlike strain-specific antibodies,those antibodies are capable of neutralizing multiple antigenicallydistinct viruses, and hence inducing such antibodies has been a focus ofnext generation universal vaccine development [Nabel, G. J. et al. NatMed 16, 1389-1391 (2010)]. However, robustly eliciting these antibodieswith such heterologous neutralizing profile by vaccination has beendifficult [Steel, J. et al. M Bio 1, e0018 (2010); Wang, T. T. et al.PLoS Pathog 6, e1000796 (2010); Wei, C. J. et al. Science 329, 1060-1064(2010)]. Removal of the immunodominant head region of HA (which containscompeting epitopes) and stabilization of the resulting stem domainthrough genetic manipulation is one potential way to improve theelicitation of these broadly neutralizing stem antibodies.

Current vaccine strategies for influenza use either a chemicallyinactivated or a live attenuated influenza virus. Both vaccines aregenerally produced in embryonated eggs which present major manufacturinglimitations due to the time consuming process and limited productioncapacity. Another more critical limitation of current vaccines is itshighly strain-specific efficacy. These challenges became glaring obviousduring emergence of the 2009 H1N1 pandemic, thus validating thenecessity for new vaccine platforms capable of overcoming theselimitations. Virus-like particles represent one of such alternativeapproaches and are currently being evaluated in clinical trials [Roldao,A. et al. Expert Rev Vaccines 9, 1149-1176 (2010); Sheridan, C. NatBiotechnol 27, 489-491 (2009)]. Instead of embryonated eggs, VLPs thatoften comprise HA, NA and matrix protein 1 (M1) can be mass-produced inmammalian or insect cell expression systems [Haynes, J. R. Expert RevVaccines 8, 435-445 (2009)]. The advantages of this approach are itsparticulate, multivalent nature and the authentic display of properlyfolded, trimeric HA spikes that faithfully mimic the infectious virion.In contrast, by the nature of its assembly, the enveloped VLPs contain asmall but finite host cell component that may present potential safety,immunogenicity challenges following repeated use of this platform [Wu,C. Y. et al. PLoS One 5, e9784 (2010)]. Moreover, the immunity inducedby the VLPs is essentially the same as current vaccines, and thus, willnot likely significantly improve both potency and breadth ofvaccine-induced protective immunity. In addition to VLPs, a recombinantHA protein has also been evaluated in humans [Treanor, J. J. et al.Vaccine 19, 1732-1737 (2001); Treanor, J. J. JAMA 297, 1577-1582(2007)], though the ability to induce protective neutralizing antibodytiters are limited. The recombinant HA proteins used in those trialswere produced in insect cells and might not form native trimerpreferentially [Stevens, J. Science 303, 1866-1870 (2004)].

Despite several alternatives to conventional influenza vaccines,advances in biotechnology in past decades have allowed engineering ofbiological materials to be exploited for the generation of novel vaccineplatforms. Ferritin, an iron storage protein found in almost all livingorganisms, is an example which has been extensively studied andengineered for a number of potential biochemical/biomedical purposes[Iwahori, K. U.S. Patent 2009/0233377 (2009); Meldrum, F. C. et al.Science 257, 522-523 (1992); Naitou, M. et al. U.S. Patent 2011/0038025(2011); Yamashita, I. Biochim Biophys Acta 1800, 846-857 (2010)],including a potential vaccine platform for displaying exogenous epitopepeptides [Carter, D. C. et al. U.S. Patent 2006/0251679 (2006); Li, C.Q. et al. Industrial Biotechnol 2, 143-147 (2006)]. Its use as a vaccineplatform is particularly interesting because of its self-assembly andmultivalent presentation of antigen which induces stronger B cellresponses than monovalent form as well as induce T-cell independentantibody responses [Bachmann, M. F. et al. Annu Rev Immunol 15, 235-270(1997); Dintzis, H. M. et al. Proc Natl Acad Sci USA 73, 3671-3675(1976)]. Further, the molecular architecture of ferritin, which consistsof 24 subunits assembling into an octahedral cage with 432 symmetry hasthe potential to display multimeric antigens on its surface.

Previous work has shown that the stem regions of Group 1 hemagglutininproteins could be modified to form to a stabilized HA stem protein, theconformation of which is very similar to the pre-fusion conformation offull-length, wild-type (wt) influenza hemagglutinin protein.Additionally, when such modified stabilized stem (SS) HA proteins werejoined to a monomeric subunit protein, such as ferritin, the resultingfusion protein formed nanoparticles, the surfaces of which displayedtrimers of the SS-HA protein. Moreover, such nanoparticles were able toelicit an immune response Group 1 influenza viruses, indicating that theSS-HA protein trimers displayed by the nanoparticles had conformationsimilar to that of wt influenza HA protein. Such constructs aredisclosed in International Patent Application No. PCT/US2015/032695, thecontent of which are incorporated herein in their entirety by reference.However, the antibodies elicited by the aforementioned nanoparticleswere more protective against Group 1 influenza viruses than they wereagainst Group 2 influenza viruses.

Thus, there remains a need for an efficacious influenza vaccine thatprovides robust protection against Group 2 influenza viruses. Further,there also remains a need for an influenza vaccine that protectsindividuals from heterologous strains of influenza virus, includingevolving seasonal and pandemic influenza virus strains of the future.The present invention meets this need by providing a novelnanoparticle-based vaccine consisting of a novel Group 2 HA stabilizedstem (SS) lacking the variable immunodominant head region, fused to thesurface of nanoparticles, resulting in an influenza vaccine that iseasily manufactured, potent, and elicits antibodies that are broadlyheterosubtypic protective.

SUMMARY OF THE INVENTION

Accordingly, this disclosure provides recombinant proteins comprising aGroup 2 influenza hemagglutinin (HA) protein, wherein the amino acidsequence of the head region is replaced with a linker comprising lessthan 5 contiguous amino acids from the head region of an influenza HAprotein. Following administration of these recombinant proteins to amammal, these recombinant proteins elicit an immune response to a Group2 influenza HA protein in the mammal.

The recombinant proteins may comprise a first amino acid sequence fromthe stem region of a Group 2 influenza virus hemagglutinin (HA) protein,and a second amino acid sequence from the stem region of a Group 2influenza virus hemagglutinin (HA) protein, wherein the first and secondamino acid sequences are covalently joined by the linker sequence, andwherein the first amino acid sequence comprises at least 20 contiguousamino acid residues from the amino acid sequence upstream of theamino-terminal end of the head region sequence, and wherein the secondamino acid sequence comprises at least 20 contiguous amino acid residuesfrom the amino acid sequence downstream of the carboxyl-terminal end ofthe head region sequence. In this recombinant protein construct, thefirst amino acid sequence may comprise at least 20 contiguous aminoacids from the upstream polypeptide sequence immediately adjacent to theamino terminal end of the head region. Alternatively or additionally,the first amino acid sequence may comprise at least 20 contiguous aminoacids from SEQ ID NO:27, SEQ ID NO: 28 or SEQ ID NO: 29. Alternativelyor additionally, the second amino acid sequence may comprise at least 20contiguous amino acids from the downstream polypeptide sequenceimmediately adjacent to the carboxyl-terminal end of the head region.Alternatively or additionally, the first amino acid sequence maycomprise at least 20 contiguous amino acids from SEQ ID NO: 31, SEQ IDNO:32 or SEQ ID NO:33.

The recombinant proteins may comprise an amino-terminal end of helix C(i.e., the membrane distal end of helix C) that is joined to the headregion sequence modified to contain a first cysteine amino acid, and alinker sequence comprising a second cysteine amino acid such that thefirst and second cysteine form a disulfide bond.

The recombinant proteins may comprise an inter-helix region (i.e., theamino acid sequence connecting the N-terminal end of helix C to thecarboxyl-terminal end of helix A (i.e., the membrane distal end of helixA)) that is modified so that the three-dimensional structure of therecombinant HA stem protein approximates the three-dimensional structureof the HA stem region in a native Group 2 HA protein. The recombinantproteins may comprise an amino acid linker sequence that is less thaneight amino acids in length, and replaces the inter-helix region.

The recombinant proteins may comprise a membrane distal end of helix Athat is extended by the addition of amino acids.

The recombinant proteins may comprise a third amino acid linker that isjoined to the carboxyl-terminus of the amino acid sequence forming helixA and forms a helix that extends the length of helix A. The distal endof helix C may be linked to the carboxyl end of the third linker by thelinker peptide. The linker peptide is preferably less than eight aminoacids in length.

These recombinant proteins may comprise one or more mutations thatincrease the stability of the protein. These stabilizing mutations arepreferably located in the amino acid sequences forming at least one ofhelix A and helix C.

These recombinant proteins may be joined to a monomeric subunit fromferritin or lumazine synthase.

Exemplary recombinant proteins of this disclosure may comprise an aminoacid sequence that is at least 80% identical, or at least 85% identical,or at least 90% identical, or at least 95% identical, or at least 97%identical, or at least 99% identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 47-159.

Exemplary recombinant proteins of this disclosure may comprise an aminoacid sequence selected from the group consisting of SEQ ID NOs: 47-159.

This disclosure also provides a nanoparticle comprising at least onerecombinant protein of this disclosure.

This disclosure also provides immunogenic compositions comprising atleast one protein that comprises an amino acid sequence at least 95%identical to these recombinant proteins. These immunogenic compositionsmay comprise a protein comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: SEQ ID NOs: 47-1598. Theseimmunogenic compositions may comprise a protein consisting of an aminoacid sequence selected from the group consisting of SEQ ID NOs:47-159.Thus, this disclosure also provides vaccine compositions comprisingthese immunogenic compositions, and an adjuvant.

This disclosure also provides methods of preventing or reducing thepathological effects of an influenza virus infection in a humancomprising administering to a human in need thereof an immunologicallyeffective dose of a vaccine composition of this disclosure.

Also provided are nucleic acids encoding the recombinant proteins ofthis disclosure. Preferably, the nucleic acid is DNA. Also provided arevectors comprising these nucleic acids. Also provided are host cellscomprising these vectors. These host cells may be bacterial cells, yeastcells, or mammalian cells. These host cells may be inactivated.

This disclosure also provides pharmaceutical compositions comprising therecombinant proteins of this disclosure. Similarly, these compositionsmay be a vaccine comprising the recombinant proteins of this disclosure,in combination with a physiologically acceptable carrier.

This disclosure also provides methods of vaccination, comprisingadministering a prophylactically or therapeutically effective amount ofa recombinant protein of this disclosure to a subject.

This disclosure also provides a method of treatment of aninfluenza-associated disease, comprising administering aprophylactically or therapeutically effective amount of a recombinantprotein of this disclosure to a subject in need thereof. Preferably, thesubject is a human.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C provide a summary of prior art. FIG. 1A is a ribbon diagramdepicting the design of full length HA-ferritin nanoparticles. FIG. 1Bis a ribbon diagram depicting the design of HA stem-ferritinnanoparticles stabilized by a HIV gp41 trimerization domain. Bothdesigns are described in detail in patent application PCT/US12/56822,which is incorporated herein by reference. FIG. 1C is a ribbon diagramdepicting the design of group 1 HA stabilized stem nanoparticlesdisclosed in PCT patent application No. PCT/US15/32695, which isincorporated herein by reference.

FIG. 2 depicts the creation of self-assembling group 2 HA stemnanoparticles. Ribbon diagrams depict (from left to right) the design ofgroup 2 HA stabilized stem nanoparticles. The head region of one HAmonomer is represented in dark gray. The stem region of that samemonomer is shown in a medium grey. The other two monomers are shown inlight gray.

FIGS. 3A and 3B show mutations in H3N2 design 231 that enable theformation of group 2 HA stabilized stem nanoparticles. FIG. 3A is aribbon diagram depicting a model of a group 2 H3N2 stabilized HA stemtrimer based on PDB ID 2YP2 . . . . Regions of mutations in the helicesare shown in dark gray. FIG. 3B shows the sequence of H3 design #231(SEQ ID NO: 47; based on the HA stem of A/Denmark35/2005 H3N2, GenBankABU92694). Mutations made to the sequence are boxed. For reference, theC-terminal SGG linker is bolded, the C-terminal ferritin is underlinedand a Asn to Gln ferritin mutation to remove an N-linked glycan isbolded.

FIGS. 4A-4D show mutations in H3N2 design 231 in the loop that replacesthe HA1 head. FIG. 4A shows a ribbon diagram depicting a model of agroup 2 H3N2 stabilized HA stem trimer based on PDB ID 2YP2. The sevenmutations in the loop that replaces the HA1 head region, and theadditional cysteine in helix C that forms a disulfide with theaforementioned loop are indicated. All other mutations in the helixregions are shown in dark grey. FIG. 4B depicts the mutated loop(indicated as replacing the head region in FIG. 4A) with side chainsrepresented by stick models. FIG. 4C shows variants of the loopsequence. The sequences are TELVFPGCGVLKL (residues 56-68 of SEQ ID NO:47), TELVFPGCVLKL (residues 56-67 of SEQ ID NO: 51), TELVFPCGVLKL(residues 56-67 of SEQ ID NO: 52), TELVFPNCGVLKL (residues 56-68 of SEQID NO: 71), and TELCFNGICLKL (residues 56-67 of SEQ ID NO: 48). FIG. 4Dshows the sequence of H3 design #231 (SEQ ID NO: 47). The mutations inthe head and helix regions, which are illustrated in FIGS. 4A-4C, areboxed. For reference, the C-terminal SGG linker is bolded, theC-terminal ferritin is underlined and a Asn to Gln ferritin mutation toremove an N-linked glycan is bolded.

FIGS. 5A-5C show mutations in H3N2, design 231, in the loop thatconnects HA2 helices A and C. FIG. 5A is a ribbon diagram depicting amodel of a group 2 H3N2 stabilized HA stem trimer based on PDB ID 2YP2.The four residues that connect HA2 helices A and C are indicated.Mutations in the helices are in dark grey. FIG. 5B shows a close-up ofthe loop (indicated region in FIG. 5A) with side chains represented bystick models. FIG. 5C shows the sequence of H3 design #231 (SEQ ID NO:47). The mutations in the helices, and the amino acids making up theshort linker, which are illustrated in FIGS. 5A and 5B, are boxed. Forreference, the C-terminal SGG linker is bolded, the C-terminal ferritinis underlined and a Asn to Gln ferritin mutation to remove an N-linkedglycan is bolded.

FIGS. 6A-6C show mutations in H3N2, design 231, in the C-terminalextension of helix A. FIG. 6A shows a ribbon diagram depicting a modelof a group 2 H3N2 stabilized HA stem trimer based on PDB ID 2YP2. Thefive-residue extension of helix A is indicated. Mutations in the helicesare in dark grey. FIG. 6B shows a close-up of the helical extension(also indicated in FIG. 6A) with side chains represented by stickmodels. FIG. 6C shows the sequence of H3 design #231 (SEQ ID NO: 47).Mutations in the helices, and the acids making up the five residueextension, are boxed. For reference, the C-terminal SGG linker isbolded, the C-terminal ferritin is underlined and a Asn to Gln ferritinmutation to remove an N-linked glycan is bolded.

FIGS. 7A and 7B show cavity-filling mutations in H3N2 design 231. FIG.7A shows a ribbon diagram depicting a model of a group 2 H3N2 stabilizedHA stem trimer based on PDB ID 2YP2. The seven cavity-filling mutationsare in dark grey with side chains represented by stick models. FIG. 7Bshows the sequence of H3 design #231 (SEQ ID NO: 47). Mutations to thehelices and head region are boxed. For reference, the C-terminal SGGlinker is bolded, the C-terminal ferritin is underlined and a Asn to Glnferritin mutation to remove an N-linked glycan is bolded.

FIGS. 8A and 8B show the expression and characterization of H3stabilized stem ferritin nanoparticle 231 (H3-SS-np_231). FIG. 8A showsa gel filtration elution profile for H3-SS-np_231 with a single peak atthe expected elution volume. The expression yield for H3-SS-np_231 fromExpi293 cells after gel filtration was 7.7 mg/L. FIG. 8B shows negativestain electron microscopy 2D class averages of H3-SS-np_231 revealingthe formation of particles with a visible arrangement of HA stemsprojecting from hollow spheres.

FIGS. 9A and 9B show HA stem antibody recognition of H3-SS-np_231. FIG.9A lists the EC₅₀ values from a kinetic ELISA H3-SS-np_231 recognitionassay by three HA stem antibodies. The values for the recognition ofH1-SS-np are also shown as a control. In both cases the nanoparticle wasimmobilized on the plate. FIG. 9B shows biolayer interferometry (BLI,from Octet) binding curves for CT149 recognition of H3-SS-np_231 (upperpanel) and BLI kinetic constants for HA stem antibodies CT149 and CR9114(lower panel).

FIGS. 10A-10E show gel filtration profiles for five variants ofH3-SS-np_231. Gel filtration Superose 6 10/30 profiles for H3-SS-np_231variants, 249 (FIG. 10A), 256 (FIG. 10B), 258 (FIG. 10C), 262 (FIG. 10D)and 264 (FIG. 10E). In each case a single peak was eluted at a volume ofapproximately 14.5 mls. The final yields from Expi293 cells after gelfiltration were 6-8 mg/L of culture.

FIGS. 11A-11F show electron microscopy of H3-SS-np nanoparticlesvariants. Negative stain electron microscopy 2D class averages ofH3-SS-np variants revealing the formation of particles with a visiblearrangement of HA stems projecting from hollow spheres. Images for theH3-SS-np_231 particle (upper left panel) are shown as a positivecontrol.

FIGS. 12A-12D show kinetic ELISA results for five variants ofH3-SS-np_231. FIGS. 12A-12C show the kinetic ELISA curves for FI6 (FIG.12A), CT149 (FIG. 12B), and CR8020 (FIG. 12C) recognition ofH3-SS-np_231 variants 249, 256, 258, 262 and 264. FIG. 12D lists theEC₅₀ values from the curves in FIGS. 12A-12C shown.

FIGS. 13A and 13B show kinetic ELISA results for H3-SS-np variants235-295. FIG. 13A lists ELISA titers showing recognition of designs235-265 by broadly neutralizing HA stem antibodies FI6, CT149 and D25(negative control). FIG. 13B lists ELISA titers showing recognition ofdesigns 266-296 by D25 and CT149. Supernatants from HEK293T cellsexpressing design immunogens were plated and detected by aboveantibodies.

FIG. 14 shows dynamic scanning calorimetry (DSC) plots for H3-SS-np(#231) and five variants. Plots of heat capacity (Cp) versus temperaturedepicts melting transitions for each protein. The earliest meltingpoints (TMs) for each design are noted. The design number is shown foreach in parentheses. In this diagram, the Cp values on the Y-axis areshown with an arbitrary scale.

FIGS. 15A & 15B show immune responses of H3-SS-np-immunized mice togroup 2 HAs. ELISA antibody endpoint titers of sera from BALB/c mice(n=10) immunized 3× with six different versions of SAS-adjuvantedH3-SS-np to plated A/Hong Kong/1/1968 (H3N2) HA (FIG. 15A) andA/Anhui/1/2013 (H7N9) (FIG. 15B). Sera from mice immunized with emptyferritin nanoparticle alone serves as a negative control. Geometric meantiters are shown by horizontal lines. Dark gray shading indicates theaverage titer for the negative control and light gray shading indicatesthe region up to four times the average titer of the negative control.Statistical analysis was performed using a two-tailed Student's t-test;*P<0.05, **P<0.01,****P<0.0001.

FIGS. 16A-16D show immune responses of H3-SS-np-immunized mice to group1 HAs. ELISA antibody endpoint titers of sera from BALB/c mice (n=10)immunized 3× with six different versions of SAS-adjuvanted H3-SS-np toplated A/New Caledonia/20/1999 (H1N1) HA (FIG. 16A), A/Canada/720/2005(H2N2) (FIG. 16B), A/Hong Kong/1074/1999 (H9N2) (FIG. 16C) andA/Vietnam/1203/2004 (H5N1) (FIG. 16D). Sera from mice immunized withempty ferritin nanoparticle alone serves as a negative control.Geometric mean titers are shown by horizontal lines. Dark gray shadingindicates the average titer for the negative control and light grayshading indicates the region up to four times the average titer of thenegative control.

FIG. 17 shows the sequence for H3-SS #231 fused to the N-terminus ofAquifex aeolicus lumazine synthase (LS) 60-mer icosahedral nanoparticles(SEQ ID NO: 83). Mutations for H3-SS-np_231 are boxed. The six residuelinker connecting H3-SS #231 to LS and a single LS mutation (N102D)deleting an N-linked glycan in LS is bolded. The C-terminal LS isunderlined.

FIGS. 18A-18F are gel filtration profiles for six variants of H3-LS-np.A-F Gel filtration Superose 6 10/30 profiles for H3-SS-LS-np variants 01(FIG. 18A), 02 (FIG. 18B), 03 (FIG. 18C), 04 (FIG. 18D), 06 (FIG. 18E)and 07 (FIG. 18F). In each case, except H3-SS-LS-04, a single peak waseluted. The final yields from Expi293 cells after gel filtration were1-2 mg/L of culture.

FIGS. 19A-19B show ELISA results for four variants of H3-LS-np. FIGS.19A and 19B show the ELISA curves for HA stem antibodies CT149 (FIG.19A) and CR8020 (FIG. 19B) recognition of H3-SS-LS-np variants 01, 02,03 and 04. The EC₅₀ values from the curves are shown below each plot.

FIG. 20 is a dynamic scanning calorimetry (DSC) plot for three H3-SS-LSvariants. Plots of heat capacity (Cp) versus temperature depicts meltingtransitions for each protein. The earliest melting points (TMs) for eachdesign are noted and color-coded to match the associated curve. Thedesign number is shown for each in parentheses. In this diagram, the Cpvalues on the Y-axis are shown with an arbitrary scale.

FIGS. 21A-21D show immune responses of H3-SS-LS-np-immunized mice todiverse HAs. ELISA antibody endpoint titers of sera from BALB/c mice(n=5) immunized 3× with four different versions of SAS-adjuvantedH3-SS-LS-np to plated A/New Caledonia/20/1999 (H1N1) HA (FIG. 21A),A/Vietnam/1203/2004 (H5N1) (FIG. 21B), A/Hong Kong/1/1968 (H3N2) (FIG.21C) and A/Anhui/1/2013 (H7N9) (FIG. 21D). Sera from mice immunized withempty ferritin nanoparticle alone and H3-SS-np (#231) serve as acontrols. Geometric mean titers are shown by horizontal lines.

FIGS. 22A and 22B show neutralizing sera responses ofH3-SS-LS-np-immunized mice to H3N2 and H7N9. Pseudovirus neutralizationtiters of sera from BALB/c mice (n=5) immunized 3× with four differentversions of SAS-adjuvanted H3-SS-LS-np. FIG. 22A shows neutralization ofA/Anhui/1/2013 (H7N9). FIG. 21B shows neutralization ofA/Wisconsin/67/2005 (H3N2). Sera from mice immunized with empty ferritinnanoparticle, H1-SS-np and H3-SS-np (#231) serve as controls. Geometricmean titers are shown by horizontal lines. Horizontal dotted linesindicate the baseline titer of 50.

FIG. 23 shows the sequence locations of the 25 mutations enable theformation of group 2 H7 HA stabilized stem nanoparticles. The sequencefor H7-SS-np_16 (SEQ ID NO: 92; based on A/Anhui/1/2013 (H7N9) HA,GenBank accession YP_009118475.1) is shown with H3 #231 mutations boxed.New H7 mutations are indicated with asterisks (two residues mutated tomatch H3N2 HA). For reference, the C-terminal SGG linker is bolded, theC-terminal ferritin is underlined and a Asn to Gln ferritin mutation toremove an N-linked glycan is bolded.

FIGS. 24A-24F show the purification of H7-SS-np variants. Gel filtrationSuperose 6 10/30 profiles for H7-SS-np variants 16 (FIG. 24A), 18 (FIG.24B), 20 (FIG. 24C), 21 (FIG. 24D), 23 (FIG. 24E) and 26 (FIG. 24F)after GNA lectin affinity chromatography. The final yields from Expi293cells after gel filtration were 5-10 mg/L of culture.

FIG. 25 shows electron microscopy of H7-SS-np. Negative stain electronmicroscopy 2D class averages of H7-SS-np variants revealing theformation of particles with a visible arrangement of HA stems projectingfrom hollow spheres. Images for an H1-SS-np particle (upper left panel)are shown as a positive control.

FIGS. 26A-26D show kinetic ELISA results for variants of H7-SS-np. FIGS.26A-26C show the kinetic ELISA curves for FI6 (FIG. 26A), CT149 (FIG.26B) and CR8020 (FIG. 26C) recognition of H7-SS-np variants 16, 18, 20,21, 23, 25, 26 and an H1-SS-np positive control. FIG. 26D lists the EC₅₀values from the curves in FIGS. 26A-26C shown. ND, not determined.

FIG. 27 shows HA stem antibody recognition of H7-SS-np. Biolayerinterferometry binding curves for CT149 recognition of six H7-SS-npvariants (FIG. 27A: H7-SS-16; FIG. 27B: H7-SS-18; FIG. 27C: H7-SS-21;FIG. 27D: H7-SS-23; FIG. 27E: H7-SS-25; FIG. 27F: H7-SS-26) are shownwith the kinetic constants listed to the right of each curve set.Nanoparticles were immobilized to the sensor tip by amine coupling andincubated in various concentrations of antibody Fabs.

FIG. 28 shows dynamic scanning calorimetry (DSC) plots for sevenH7-SS-np variants. Plots of heat capacity (Cp) versus temperaturedepicts melting transitions for each protein. The earliest meltingpoints (TMs) for each protein are noted and color-coded to match theassociated curve. The H7-SS-np design number is shown for each inparentheses. In this diagram, the Cp values on the Y-axis are shown withan arbitrary scale.

FIGS. 29A-29D show immune responses of H7-SS-np-immunized mice todiverse HAs. ELISA antibody endpoint titers of sera from BALB/c mice(n=5) immunized 3× with six different versions of SAS-adjuvantedH7-SS-np to plated A/New Caledonia/20/1999 (H1N1) HA (FIG. 29A),A/Vietnam/1203/2004 (H5N1) (FIG. 29B), A/Hong Kong/1/1968 (H3N2) (FIG.29C) and A/Anhui/1/2013 (H7N9) (FIG. 29D). Sera from mice immunized withempty ferritin nanoparticle, H1-SS-np and H3-SS-np (#231) serve ascontrols. Geometric mean titers are shown by horizontal lines.Horizontal dotted lines indicate the baseline titer of 50.

FIGS. 30A and 30B show neutralizing sera responses of H7-SS-np-immunizedmice to H3N2 and H7N9. Pseudovirus neutralization titers of sera fromBALB/c mice (n=5) immunized 3× with six different versions ofSAS-adjuvanted H7-SS-np. FIG. 30A shows neutralization to A/Anhui/1/2013(H7N9). FIG. 30B shows neutralization of A/Wisconsin/67/2005 (H3N2).Sera from mice immunized with empty ferritin nanoparticle, H1-SS-np andH3-SS-np (#231) serve as controls. Geometric mean titers are shown byhorizontal lines. Horizontal dotted lines indicate the baseline titer of50.

FIG. 31 shows the sequence of four different examples of proteinconstructs of the invention, based on the sequence of the influenzasubtype 10 HA (H10) protein. Mutations made to the influenza HAsequences are boxed. For reference, the C-terminal SGG linker is bolded,and the C-terminal ferritin sequence is underlined.

FIGS. 32A-32E show gel filtration Superdex 200 10/30 profiles for H10ssFvariants 1 (FIG. 32A), 2 (FIG. 32B) 3 (FIG. 32C), 4 (FIG. 32D) and 5(FIG. 32E). In each case a single peak was eluted at a volume ofapproximately 12.5 mls. The final yields from Expi293 cells after gelfiltration were 6-8 mg/L of culture.

FIG. 33. Electron microscopy of H10ssF nanoparticles variants. Negativestain electron microscopy 2D class averages of H10ssF variants revealingthe formation of particles with a visible arrangement of HA stemsprojecting from hollow spheres.

FIGS. 34A-34 D show kinetic ELISA results for H10ssF variants 2-5. FIGS.A-C. show ELISA curves. FIG. D. shows IC50 values calculated from thecurves. Supernatants from HEK293T cells expressing design immunogenswere plated and detected by above antibodies

FIGS. 35A & 35B show immune responses of H10ssF-immunized mice to group2 HAs. ELISA antibody endpoint titers of sera from BALB/c mice (n=10)immunized 3× with five different versions of SSAS-adjuvanted H10ssF (2ug/mouse) to immobilized A/Hong Kong/1/1968 (H3N2) HA (FIG. 35A) andA/Anhui/1/2013 (H7N9) (FIG. 35B). Responses to sera from mice immunizedwith empty ferritin nanoparticle alone, H7N9 AH13 Monovalent inactivatedvaccine (MIV) or H7ssF26 serve as controls. Geometric mean titers areshown by horizontal lines. The bottom dotted line indicates the baselinetiter of 50 and the top dotted line indicates the highest valuerecorded.

FIGS. 36A-36D show the responses of H10ssF-immunized mice to a lethalH3N2 challenge. FIGS. 36A-C. shows weight loss curves for BALB/c mice(n=10) immunized with empty nanoparticles (FIG. 36A), H10ssF_4 (FIG.36B), or H10ssF_5 (FIG. 36C), and then challenged with a lethal dose ofA/Philippines/1982 (H3N2) influenza. FIG. 36D. shows survival curves forthe same mice as in A. Mice immunized with ferritin nanoparticle alone(empty np) serve as a negative control.

FIGS. 37A-37G show responses of H10ssF-immunized mice to a lethal H7N9challenge. FIG. 37A. shows survival curves for H10ssF-immunized BALB/cmice (n=10) challenged with a lethal dose of A/Shanghai/2/2013-like(H7N9) influenza. Mice immunized with ferritin nanoparticle alone (emptynp) serve as a negative control. FIG. 37B shows weight loss six dayspost challenge for the same mice as in FIG. 37A. FIGS. 37C-G show weightloss over 12 days post challenge for the same mice as in FIGS. 37A &3B7.

FIG. 38 shows the sequence of four different examples of proteinconstructs of the invention, based on the sequence of the influenzasubtype 3 HA (H3) protein. Mutations made to the influenza HA sequencesare boxed. For reference, the C-terminal SGG linker is bolded, and theC-terminal ferritin sequence is underlined. Also, a Asn to Gln ferritinmutation that removes an N-linked glycan is boxed and bolded.

FIG. 39 shows the sequence of four different examples of proteinconstructs of the invention, based on the sequence of the influenzasubtype 7 HA (H7) protein. Mutations made to the influenza HA sequencesare boxed. For reference, the C-terminal SGG linker is bolded, and theC-terminal ferritin sequence is underlined. Also, a Asn to Gln ferritinmutation that removes an N-linked glycan is boxed and bolded.

FIGS. 40A-40C shows the ability of various protein constructs of theinvention to activate B cells expressing germline-reverted 16.a.26 Bcell receptors (BCRs). The graphs show calcium flux (indicating B-cellactivation) resulting from contact of the B-cells with an anti-IgMpositive control (and no activation using H1 negative control) (FIG.40A), H3-ss-np protein constructs (FIG. 40B), H7-ss-np proteinconstructs (FIG. 40C), and H10ssF protein constructs (FIG. 40D).

FIG. 41 shows the sequence of HA portion of protein constructs thatexhibited activity in the B-cell activation assay illustrated in FIGS.40A-C. Mutations made to the influenza HA sequences are boxed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a novel vaccine for influenza virus.More specifically, the present invention relates to novel, Group 2influenza HA protein-based vaccines that elicit an immune responseagainst the stem region of the HA protein from a broad range ofinfluenza viruses. It also relates to self-assembling nanoparticles thatdisplay immunogenic portions of the pre-fusion conformation of the stemregion from the Group 2 influenza HA protein on their surface. Suchnanoparticles are useful for vaccinating individuals against influenzavirus. Accordingly, the present invention also relates to proteinconstructs for producing such nanoparticles and nucleic acid moleculesencoding such proteins. Additionally, the present invention relates tomethods of producing nanoparticles of the present invention, and methodsof using such nanoparticles to vaccinate individuals.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. For example, a nucleic acid moleculerefers to one or more nucleic acid molecules. As such, the terms “a”,“an”, “one or more” and “at least one” can be used interchangeably.Similarly, the terms “comprising”, “including” and “having” can be usedinterchangeably. It is further noted that the claims may be drafted toexclude any optional element. As such, this statement is intended toserve as antecedent basis for use of such exclusive terminology as“solely,” “only” and the like in connection with the recitation of claimelements, or use of a “negative” limitation.

For convenience, certain abbreviations can be used to refer to proteinconstructs, and portions thereof, of the invention. For example, HA canrefer to influenza hemagglutinin protein, or a portion thereof. HA-SSrefers to a stabilized stem region, or a portion of the stem region,from an influenza HA protein. Typically the HA portion of such adesignation will refer to the subtype of the hemagglutinin protein. Forexample, a stabilized stem region from a subtype 3 hemagglutinin can bereferred to as H3-SS. A protein construct comprising a HA-SS (e.g.,H3-SS) joined to an influenza HA protein transmembrane domain can bereferred to as HA-SS-TM (e.g., H3-SS-TM). A protein constructscomprising a HA-SS joined to a ferritin monomeric subunit can bereferred to as HA-SS-np. Such a designation may also be followed by anumber that indicates a particular construct containing specificalterations (e.g., H3-SS-np_231 (SEQ ID NO:47)). It should be noted thatsuch a construct can also be referred to HAssF (e.g., H3ssF_231). Incertain aspects of the invention, a HA-SS is joined to other monomericsubunits, such as, for example, lumazine synthase. Such a construct canbe referred to by the designation HA-SS-LS (e.g., H3-SS_LS-01 (SEQ IDNO:83)) or HAssL (e.g., H3ssLS-01 (SEQ ID NO:83)).

In addition to the above, unless specifically defined otherwise, thefollowing terms and phrases, which are common to the various embodimentsdisclosed herein, are defined as described below.

As used herein, a protein construct is a protein made by the hand ofman, in which the amino acid sequence of a protein is modified so thatthe resulting modified protein comprises a sequence that is not found innature. Protein constructs include protein in which two or more aminoacid sequences have been covalently joined in a way not found in nature.The amino acid sequences being joined can be related or unrelated. Asused herein, polypeptide sequences are unrelated, if their amino acidsequences are not normally found joined together via a covalent bond intheir natural environment(s) (e.g., inside a cell). For example, theamino acid sequence of a ferritin monomeric subunit, and the amino acidsequence of a Group 2 influenza HA protein are not normally found joinedtogether via a covalent bond. Thus, such sequences are consideredunrelated.

Protein constructs can also comprise related amino acid sequences. Forexample, the structure of the influenza HA protein is such that the headregion amino acid sequence is flanked on both ends by stem region aminoacid sequences. Through genetic means, it is possible to create amodified version of an HA protein by removing amino acid residues fromthe middle of the head region, while maintaining a portion of the headregion flanked by stem regions sequences. While the order of thesequences in the final molecule would remain the same, the spatialrelationship between the amino acids would differ from the naturalprotein. Thus, such a molecule would be considered a protein construct.According to the present invention, protein constructs may also bereferred to as fusion proteins.

Amino acid sequences in a protein construct can be joined directly toeach other or they can be joined using a linker. A linker, linkersequence, linker peptide, and the like, is a short (e.g., 2-20) aminoacid sequence used to connect two proteins having a desiredcharacteristic (e.g., structure, epitope, immunogenicity, activity,etc.). A linker sequence typically does not have its own activity and isusually used to connect other parts of the protein construct, therebyallowing them to assume a desired conformation. Linker sequences aretypically made from small amino acid residues and/or runs thereof, suchas, for examples, serine, alanine and glycine, although the use of otheramino acid residues is not excluded. For example, it may be desirable toinclude an amino acid that can form a covalent bond, such as a cysteineresidue, in the linker sequence.

As used herein, the term immunogenic refers to the ability of a specificprotein, or a specific region thereof, to elicit an immune response tothe specific protein, or to proteins comprising an amino acid sequencehaving a high degree of identity with the specific protein. According tothe present invention, two proteins having a high degree of identityhave amino acid sequences at least 80% identical, at least 85%identical, at least 87% identical, at least 90% identical, at least 92%identical, at least 93% identical, at least 94% identical, at least 95%identical, at least 96% identical, at least 97% identical, at least 98%identical or at least 99% identical. Methods of determining the percentidentity between two amino acid or nucleic acid sequence are known inthe art.

As used herein, an immune response to a vaccine, or nanoparticle, of thepresent invention is the development in a subject of a humoral and/or acellular immune response to a Group 2 HA protein present in the vaccine.For purposes of the present invention, a “humoral immune response”refers to an immune response mediated by antibody molecules, includingsecretory (IgA) or IgG molecules, while a “cellular immune response” isone mediated by T-lymphocytes and/or other white blood cells. Oneimportant aspect of cellular immunity involves an antigen-specificresponse by cytolytic T-cells (“CTL”s). CTLs have specificity forpeptide antigens that are presented in association with proteins encodedby the major histocompatibility complex (MHC) and expressed on thesurfaces of cells. CTLs help induce and promote the destruction ofintracellular microbes, or the lysis of cells infected with suchmicrobes. Another aspect of cellular immunity involves anantigen-specific response by helper T-cells. Helper T-cells act to helpstimulate the function, and focus the activity of, nonspecific effectorcells against cells displaying peptide antigens in association with MHCmolecules on their surface. A cellular immune response also refers tothe production of cytokines, chemokines and other such moleculesproduced by activated T-cells and/or other white blood cells, includingthose derived from CD4+ and CD8+ T-cells.

Thus, an immunological response may be one that stimulates CTLs, and/orthe production or activation of helper T-cells. The production ofchemokines and/or cytokines may also be stimulated. The vaccine may alsoelicit an antibody-mediated immune response. Hence, an immunologicalresponse may include one or more of the following effects: theproduction of antibodies (e.g., IgA or IgG) by B-cells; and/or theactivation of suppressor, cytotoxic, or helper T-cells and/or T-cellsdirected specifically to a Group 2 HA protein present in the vaccine.These responses may serve to neutralize infectivity (e.g.,antibody-dependent protection), and/or mediate antibody-complement, orantibody dependent cell cytotoxicity (ADCC) to provide protection to animmunized individual. Such responses can be determined using standardimmunoassays and neutralization assays, well known in the art.

As used herein, the term antigenic, antigenicity, and the like, refersto a protein that is bound by an antibody or a group of antibodies.Similarly, an antigenic portion of a protein is any portion that isrecognized by an antibody or a group of antibodies. According to thepresent invention, recognition of a protein by an antibody means theantibody selectively binds to the protein. As used herein, the phraseselectively binds, selective binding, and the like, refer to the abilityof an antibody to preferentially bind an HA protein as opposed tobinding proteins unrelated to HA, or non-protein components in thesample or assay. An antibody that preferentially binds HA is one thatbinds HA but does not significantly bind other molecules or componentsthat may be present in the sample or assay. Significant binding isconsidered, for example, binding of an anti-HA antibody to a non-HAmolecule with an affinity or avidity great enough to interfere with theability of the assay to detect and/or determine the level ofanti-influenza antibodies, or HA protein, in the sample. Examples ofother molecules and compounds that may be present in the sample, or theassay, include, but are not limited to, non-HA proteins, such asalbumin, lipids and carbohydrates. According to the present invention, anon-HA protein is a protein having an amino acid sequence sharing lessthan 60% identity with the sequence of an influenza HA protein disclosedherein. In some embodiments, the antibody or antibodies provide broadheterosubtypic protection. In some embodiments, the antibody orantibodies are neutralizing.

As used herein, neutralizing antibodies are antibodies that preventinfluenza virus from completing one round of replication. As definedherein, one round of replication refers the life cycle of the virus,starting with attachment of the virus to a host cell and ending withbudding of newly formed virus from the host cell. This life cycleincludes, but is not limited to, the steps of attaching to a cell,entering a cell, cleavage and rearrangement of the HA protein, fusion ofthe viral membrane with the endosomal membrane, release of viralribonucleoproteins into the cytoplasm, formation of new viral particlesand budding of viral particles from the host cell membrane. According tothe present invention, a neutralizing antibody is one that inhibits oneor more such steps.

As used herein, broadly neutralizing antibodies are antibodies thatneutralize more than one type, subtype and/or strain of influenza virus.For example, broadly neutralizing antibodies elicited against an HAprotein from a Type A influenza virus may neutralize a Type B or Type Cvirus. As a further example, broadly neutralizing antibodies elicitedagainst an HA protein from Group 2 influenza virus may neutralize aGroup 1 virus. As an additional example, broadly neutralizing antibodieselicited against an HA protein from one sub-type or strain of virus, mayneutralize another sub-type or strain of virus. For example, broadlyneutralizing antibodies elicited against an HA protein from an H3influenza virus may neutralize viruses from one or more sub-typesselected from the group consisting of H1, H2, H4, H5, H6, H7, H8, H8,H10, H11, H12, H13, H14, H15, H16, H17 or H18.

According to the present invention all nomenclature used to classifyinfluenza virus is that commonly used by those skilled in the art. Thus,a Type, or Group, of influenza virus refers to influenza Type A,influenza Type B or influenza type C. It is understood by those skilledin the art that the designation of a virus as a specific Type relates tosequence difference in the respective M1 (matrix) protein or NP(nucleoprotein). Type A influenza viruses are further divided into Group1 and Group 2. These Groups are further divided into subtypes, whichrefers to classification of a virus based on the sequence of its HAprotein. Examples of current commonly recognized subtypes are H1, H2,H3, H4, H5, H6, H7, H8, H8, H10, H11, H12, H13, H14, H15, H16, H17 orH18. Group 1 influenza subtypes are H1, H2, H5, H6, H8, H9, H11, H12,H13, H16, H17 and H18. Group 2 influenza subtypes are H3, H4, H7, H10,H14, and H15. Finally, the term strain refers to viruses within asubtype that differ from one another in that they have small, geneticvariations in their genome.

As used herein, an influenza hemagglutinin protein, or HA protein,refers to a full-length influenza hemagglutinin protein or any portionthereof, that is useful for producing protein constructs andnanoparticles of the invention or that are capable of eliciting animmune response. Preferred HA proteins are those that are capable offorming a trimer. An epitope of a full-length influenza HA proteinrefers to a portion of such protein that can elicit an antibody responseagainst the homologous influenza strain, i.e., a strain from which theHA is derived. In some embodiments, such an epitope can also elicit anantibody response against a heterologous influenza strain, i.e., astrain having an HA that is not identical to that of the HA of theimmunogen. In some embodiments, the epitope elicits a broadlyheterosubtypic protective response. In some embodiments, the epitopeelicits neutralizing antibodies.

As used herein, a variant refers to a protein, or nucleic acid molecule,the sequence of which is similar, but not identical to, a referencesequence, wherein the activity of the variant protein (or the proteinencoded by the variant nucleic acid molecule) is not significantlyaltered. These variations in sequence can be naturally occurringvariations or they can be engineered through the use of geneticengineering technique known to those skilled in the art. Examples ofsuch techniques are found in Sambrook J, Fritsch E F, Maniatis T et al.,in Molecular Cloning—A Laboratory Manual, 2nd Edition, Cold SpringHarbor Laboratory Press, 1989, pp. 9.31-9.57), or in Current Protocolsin Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, bothof which are incorporated herein by reference in their entirety.

With regard to variants, any type of alteration in the amino acid, ornucleic acid, sequence is permissible so long as the resulting variantprotein retains the ability to elicit neutralizing or non-neutralizingantibodies against an influenza virus. Examples of such variationsinclude, but are not limited to, deletions, insertions, substitutionsand combinations thereof. For example, with regard to proteins, it iswell understood by those skilled in the art that one or more (e.g., 2,3, 4, 5, 6, 7, 8, 9 or 10), amino acids can often be removed from theamino and/or carboxyl terminal ends of a protein without significantlyaffecting the activity of that protein. Similarly, one or more (e.g., 2,3, 4, 5, 6, 7, 8, 9 or 10) amino acids can often be inserted into aprotein without significantly affecting the activity of the protein. Invariants into which insertions have been made, the inserted amino acidsmay be referred to by referencing the amino acid residue after which theinsertion was made. For example, an insertion of four amino acidresidues after amino acid residue 402 could be referred to as 402 a-402d. Moreover, if one of those inserted amino acids are later substitutedwith another amino acid, such a change can be referred to by referenceto the letter position. For example, substitution of an inserted glycine(in the further position of the insert) with a threonine can be referredto as S402dT.

As noted, variant proteins of the present invention can contain aminoacid substitutions relative to the influenza HA proteins disclosedherein. Any amino acid substitution is permissible so long as theactivity of the protein is not significantly affected. In this regard,it is appreciated in the art that amino acids can be classified intogroups based on their physical properties. Examples of such groupsinclude, but are not limited to, charged amino acids, uncharged aminoacids, polar uncharged amino acids, and hydrophobic amino acids.Preferred variants that contain substitutions are those in which anamino acid is substituted with an amino acid from the same group. Suchsubstitutions are referred to as conservative substitutions.

Naturally occurring residues may be divided into classes based on commonside chain properties:

1) hydrophobic: Met, Ala, Val, Leu, Ile;

2) neutral hydrophilic: Cys, Ser, Thr;

3) acidic: Asp, Glu;

4) basic: Asn, Gln, His, Lys, Arg;

5) residues that influence chain orientation: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions may involve the exchange ofa member of one of these classes for a member from another class.

In making amino acid changes, the hydropathic index of amino acids maybe considered. Each amino acid has been assigned a hydropathic index onthe basis of its hydrophobicity and charge characteristics. Thehydropathic indices are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5). The importance of the hydropathicamino acid index in conferring interactive biological function on aprotein is generally understood in the art (Kyte et al., 1982, J. Mol.Biol. 157:105-31). It is known that certain amino acids may besubstituted for other amino acids having a similar hydropathic index orscore and still retain a similar biological activity. In making changesbased upon the hydropathic index, the substitution of amino acids whosehydropathic indices are within ±2 is preferred, those within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functionally equivalent protein orpeptide thereby created is intended for use in immunological invention,as in the present case. The greatest local average hydrophilicity of aprotein, as governed by the hydrophilicity of its adjacent amino acids,correlates with its immunogenicity and antigenicity, i.e., with abiological property of the protein. The following hydrophilicity valueshave been assigned to these amino acid residues: arginine (+3.0); lysine(+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); and tryptophan (−3.4). In makingchanges based upon similar hydrophilicity values, the substitution ofamino acids whose hydrophilicity values are within ±2 is preferred,those within ±1 are particularly preferred, and those within ±0.5 areeven more particularly preferred. One may also identify epitopes fromprimary amino acid sequences on the basis of hydrophilicity.

Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. For example, amino acidsubstitutions can be used to identify important residues of the HAprotein, or to increase or decrease the immunogenicity, solubility orstability of the HA proteins described herein. Exemplary amino acidsubstitutions are shown below in Table 1.

TABLE 1 Amino Acid Substitutions Original Amino Acid ExemplarySubstitutions Ala Val, Leu, Ile Arg Lys, Gln, Asn Asn Gln Asp Glu CysSer, Ala Gln Asn Glu Asp Gly Pro, Ala His Asn, Gln, Lys, Arg Ile Leu,Val, Met, Ala Leu Ile, Val, Met, Ala Lys Arg, Gln, Asn Met Leu, Phe, IlePhe Leu, Val, Ile, Ala, Tyr Pro Ala Ser Thr, Ala, Cys Thr Ser Trp Tyr,Phe Tyr Trp, Phe, Thr, Ser Val Ile, Met, Leu, Phe, Ala

As used herein, the phrase “significantly affect a protein activity”refers to a decrease in the activity of a protein by at least 10%, atleast 20%, at least 30%, at least 40% or at least 50%. With regard tothe present invention, such an activity may be measured, for example, asthe ability of a protein to elicit protective antibodies against aninfluenza virus. Such activity may be measured by measuring the titer ofsuch antibodies against influenza virus, the ability of such antibodiesto protect against influenza infection or by measuring the number oftypes, subtypes or strains neutralized by the elicited antibodies.Methods of determining antibody titers, performing protection assays andperforming virus neutralization assays are known to those skilled in theart. In addition to the activities described above, other activitiesthat may be measured include the ability to agglutinate red blood cellsand the binding affinity of the protein for a cell. Methods of measuringsuch activities are known to those skilled in the art.

The terms individual, subject, and patient are well-recognized in theart, and are herein used interchangeably to refer to any human or otheranimal susceptible to influenza infection. Examples include, but are notlimited to, humans and other primates, including non-human primates suchas chimpanzees and other apes and monkey species; farm animals such ascattle, sheep, pigs, seals, goats and horses; domestic mammals such asdogs and cats; laboratory animals including rodents such as mice, ratsand guinea pigs; birds, including domestic, wild and game birds such aschickens, turkeys and other gallinaceous birds, ducks, geese, and thelike. The terms individual, subject, and patient by themselves, do notdenote a particular age, sex, race, and the like. Thus, individuals ofany age, whether male or female, are intended to be covered by thepresent disclosure and include, but are not limited to the elderly,adults, children, babies, infants, and toddlers. Likewise, the methodsof the present invention can be applied to any race, including, forexample, Caucasian (white), African-American (black), Native American,Native Hawaiian, Hispanic, Latino, Asian, and European. An infectedsubject is a subject that is known to have influenza virus in theirbody.

As used herein, a vaccinated subject is a subject that has beenadministered a vaccine that is intended to provide a protective effectagainst an influenza virus.

As used herein, the terms exposed, exposure, and the like, indicate thesubject has come in contact with a person of animal that is known to beinfected with an influenza virus.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates, which may need to be independently confirmed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodiments arespecifically embraced by the present invention and are disclosed hereinjust as if each and every combination was individually and explicitlydisclosed. In addition, all sub-combinations are also specificallyembraced by the present invention and are disclosed herein just as ifeach and every such sub-combination was individually and explicitlydisclosed herein.

One embodiment of the present invention is a protein constructcomprising a Group 2 influenza HA protein wherein the head region of theGroup 2 influenza HA protein has been replaced with an amino acidsequence comprising less than 5 contiguous amino acid residues from thehead region of an influenza HA protein. As used herein, a Group 2 HAprotein, refers to a full-length influenza HA protein from a Group 2influenza virus, or any portion/portions and/or variants thereof, thatis/are useful for producing protein constructs and nanoparticles of theinvention. Accordingly, the present invention is drawn to molecules thatare capable of eliciting an immune response to the stem region of aGroup 2 influenza HA protein. In some embodiments, the sequence of theHA protein construct has been further altered (i.e., mutated) tostabilize the stem region of the protein in a form that can be presentedto the immune system. Examples of Group 2 influenza HA proteins usefulfor practicing the invention, and protein constructs made therefrom, areshown in Table 2, below.

TABLE 2 PCT SEQ ID NO Comments Monomeric Subunit Proteins 1Amino acid sequence of ferritin monomeric subunit protein from H. pylori,MLSDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLA DQYVKGIAKSRKSGS 2amino acids 4-168 from SEQ ID NO: 2; Asn19 has been replaced with Gln,DIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQY VKGIAKSRKSGS 3Amino acid sequence of lumazine synthase from aquifex aeolicus,MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSL R FULL LENGTH HA 4amino acid sequence of hemagglutinin protein from influenza A virus(A/Denmark/35/2005 (H3N2)); GenBank: ABU92694.1 5amino acid sequence of hemagglutinin protein from influenza A virus(A/Bangladesh/558/2012 (H3N2)); Accession: AJB43527.1 6amino acid sequence of hemagglutinin protein from influenza A virus(A/Sao Paulo/89403/2010 (H3N2)); Accession: AET10116.1 7amino acid sequence of hemagglutinin protein from influenza A virus(A/Bangladesh/541/2012 (H3N2)); Accession: AJB43525.1 8amino acid sequence of hemagglutinin protein from influenza A virus(A/Bangladesh/542/2012(H3N2)); Accession: AJB43524.1 9amino acid sequence of hemagglutinin protein from influenza A virus(A/Tocantins/979/2010 (H3N2)); Accession: AET10115.1 10amino acid sequence of hemagglutinin protein from influenza A virus(A/Tunisia/17332/2011 (H3N2)); Accession: AFV68725.1 11amino acid sequence of hemagglutinin protein from influenza A virus(A/Norway/88/2003 (H3N2)); Accession: ABR14669.1 12amino acid sequence of hemagglutinin protein from influenza A virus(A/Japan/AF2844/2012 (H3N2)); Accession: AFH57071.1 13amino acid sequence of hemagglutinin protein from influenza A virus(A/Texas/2977/2012(H3N2)); Accession: AFM45466.1 14amino acid sequence of hemagglutinin protein from influenza A virus(A/North Carolina/AF2716/2011 (H3N2)); Accession: ADY05375.1 15amino acid sequence of hemagglutinin protein from influenza A virus(A/Norway/70/2005 (H3N2)); Accession: ABI22080.1 16amino acid sequence of hemagglutinin protein from influenza A virus(A/duck/Chiba/24-203-44/2012 (H7N1)); Accession: BAN16716.1 17amino acid sequence of hemagglutinin protein from influenza A virus(A/chicken/Germany/2003 (H7N7)); Accession: CAG28959.1 18amino acid sequence of hemagglutinin protein from influenza A virus(A/chicken/Italy/444/1999 (H7N1)); Accession: CAG28956.1 19amino acid sequence of hemagglutinin protein from influenza A virus(A/mallard/Italy/4810-7/2004 (H7N7)); Accession: ABG57092.1 20amino acid sequence of hemagglutinin protein from influenza A virus(A/Anhui/DEWH72-03/2013 (H7N9)); Accession: AHZ39710.1 21amino acid sequence of hemagglutinin protein from influenza A virus(A/Shanghai/JS01/2013 (H7N9)); Accession: AGW82612.1 22amino acid sequence of hemagglutinin protein from influenza A virus(A/Guangdong/02/2013 (H791)); Accession: AHD25003.1 23amino acid sequence of hemagglutinin protein from influenza A virus(A/Shenzhen/SP44/2014 (H7N9)); Accession: AJJ91957.1 24amino acid sequence of hemagglutinin protein from influenza A virus(A/Beijing/3/2013 (H7N9)); Accession: AHM24224.1 25amino acid sequence of hemagglutinin protein from influenza A virus(A/Hong Kong/470129/2013 (H7N9)); Accession: AHF20528.1 26amino acid sequence of hemagglutinin protein from influenza A virus(A/Jiangxi/IPB13/2013 (H10N8; Accession: AHK10761.1) Flanking Sequences27Amino acid sequence flanking amino-terminal end of head region from influenzavirus A (Denmark/35/2005 (H3N2))-full(aa1-59)MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIE VTNATELV 28Amino acid sequence flanking amino-terminal end of head region from influenzavirus A (Denmark/35/2005 (H3N2))-partial (40 aa′s)PGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELV 29Amino acid sequence flanking amino-terminal end of head region from influenzavirus A (Denmark/35/2005 (H3N2))-partial (25 aa′s)AVPNGTIVKTITNDQIEVTNATELV 30Amino acid sequence of stem region flanking carboxyl-terminal end of head regionfrom influenza virus A (Denmark/35/2005 (H3N2))LKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGIGQAADLKSTQAAINQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKVDLWSYNAELLVALENQHTIDLTDSEMNKLFERTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIK 31Amino acid sequence of stem region flanking carboxyl-terminal end of head regionfrom influenza virus A (Denmark/35/2005 (H3N2))-partial-66 aa′s)LKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGIGQA ADLKSTQAAINQING 32Amino acid sequence of stem region flanking carboxyl-terminal end of head regionfrom influenza virus A (Denmark/35/2005 (H3N2))-partial-50 aa′s)LKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGIGQ 33Amino acid sequence of stem region flanking carboxyl-terminal end of head regionfrom influenza virus A (Denmark/35/2005 (H3N2))-partial-25 aa′s)LKLATGMRNVPEKQTRGIFGAIAGF Linker Sequences 34 VFPGCGV-head linker 35CFNGIC-head linker 36 Helix A extension sequence-ALMAQ 37Helix A extension sequence-ELMEQ 38Inter-helix region-GKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKVDLW 39Inter-helix linker-GGPDHead region carboxyl flank (inter-helix region replaced with linker) 40DLKSTQAAINQINGKLNRLIALMAQGGPDSYNAELLVALENQHTIDLTD 41NSEGIGQAADLKSTQAAINQINGKLNRLIALMAQGGPDSYNAELLVALE NQHTIDLTDSEMNKLFERT 42NSEGIGQAADLKSTQAAINQINGKLNRLIALMAQGGPDSYNAELLVALENQHTIDLTDSEMNKLFERTKKQLRENAEDMGNGCFKIYH 43LKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGIGQAADLKSTQAAINQINGKLNRLIALMAQGGPDSYNAELLVALENQHTIDLTDSEMNKLFERTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYD HDVYRDEALNNRFQIKInter-helix carboxyl flank-goes all the way to end of stem;does not include TM domain 44 SYNAELLVALENQHTIDLTDSEMNKLFERTKKQLRENAEDMG45 SYNAELLVALENQHTIDLTDSEMNKLFERTKKQLRENAEDMGNGCFKIY HKCDNACIGSIRN 46SYNAELLVALENQHTIDLTDSEMNKLFERTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKProtein Constructs With HA Joined to Monomeric Subunit 47Amino acid sequence of H3-SS-np_231; (H3ssF_231) 48Amino acid sequence of H3-SS-np_249; (H3ssF_249) 49Amino acid sequence of H3-SS-np_256; (H3ssF_256) 50Amino acid sequence of H3-SS-np_258; (H3ssF_258) 51Amino acid sequence of H3-SS-np_262; (H3ssF_262) 52Amino acid sequence of H3-SS-np_264; (H3ssF_264) 53Amino acid sequence of H3-SS-np_265; (H3ssF_265) 54Amino acid sequence of H3-SS-np_266; (H3ssF_266) 55Amino acid sequence of H3-SS-np_267; (H3ssF_267) 56Amino acid sequence of H3-SS-np_268; (H3ssF_268) 57Amino acid sequence of H3-SS-np_269; (H3ssF_269) 58Amino acid sequence of H3-SS-np_270; (H3ssF_270) 59Amino acid sequence of H3-SS-np_271; (H3ssF_271) 60Amino acid sequence of H3-SS-np_272; (H3ssF_272) 61Amino acid sequence of H3-SS-np_279; (H3ssF_279) 62Amino acid sequence of H3-SS-np_281; (H3ssF_281) 63Amino acid sequence of H3-SS-np_287; (H3ssF_287) 64Amino acid sequence of H3-SS-np_288; (H3ssF_288) 65Amino acid sequence of H3-SS-np_289; (H3ssF_289) 66Amino acid sequence of H3-SS-np_291; (H3ssF_291) 67Amino acid sequence of H3-SS-np_292; (H3ssF_292) 68Amino acid sequence of H3-SS-np_293; (H3ssF_293) 69Amino acid sequence of H3-SS-np_294; (H3ssF_294) 70Amino acid sequence of H3-SS-np_295; (H3ssF_295) 71Amino acid sequence of H3-SS-np_296 (based on H7 #21); (H3ssF_296) 72Amino acid sequence of H3-SS-np_297 (based on H7 #23); (H3ssF_297) 73Amino acid sequence of H3-SS-np_298 (based on #249 and H7 #23); (H3ssF_298)74Amino acid sequence of H3-SS-np_299 (based on #249 and #258); (H3ssF_299)75 Amino acid sequence of H3-SS-np_231_HK68; (H3ssF_231_HK68) 76Amino acid sequence of H3-SS-np_231_BK79; (H3ssF_231_BK79) 77Amino acid sequence of H3-SS-np_231_Wyo03; (H3ssF_231_Wyo03) 78Amino acid sequence of H3-SS-np_231_Switz13; (H3ssF_231_Switz13) 79Amino acid sequence of H3-SS-np_262_HK68; (H3ssF_262_HK68) 80Amino acid sequence of H3-SS-np_262_BK79; (H3ssF_262_BK79) 81Amino acid sequence of H3-SS-np_262_Wyo03; (H3ssF_262_Wyo03) 82Amino acid sequence of H3-SS-np_262_Switz13; (H3ssF_262Switz13) 83Amino acid sequence of H3-SS_LS-01 (based on #231, N298D, Linker extension);(H3ssLS-01) 84Amino acid sequence of H3-SS_LS-02 (based on #231, M197C, I244C, N298D,linker extension, added glutamates); (H3ssLS-02) 85Amino acid sequence of H3-SS_LS-03 (based on #231, N298D, linker extension,added glutamates); (H3ssLS-03) 86Amino acid sequence of H3-SS_LS-04 (based on #231, M197, I244C, N298D, linkerextension, added glutamates); (H3ssLS-04) 87Amino acid sequence of H3-SS_LS-05 (based on #266, S300A, linker extension);(H3ssLS-05) 88Amino acid sequence of H3-SS_LS-06 (based on #266, N298D, linker extension);(H3ssLS-06) 89Amino acid sequence of H3-SS_LS-07 (based on #274, N298D, linker extension);(H3ssLS-07) 90 Amino acid sequence of H3-SS-SA_01 91Amino acid sequence of H3-SS_SA_02 92Amino acid sequence of H7-SS-np_016 (based on H3 #231); (H7ssF_016) 93Amino acid sequence of H7-SS-np_018 (based on H3 #262); (H7ssF_018) 94Amino acid sequence of H7-SS-np_020 (based on H3 #264); (H7ss_F020) 95Amino acid sequence of H7-SS-np_021 (based on a variation of H3 #231);(H7ssF_021) 96Amino acid sequence of H7-SS-np_023 (based on a variation of H3 #231);(H7ssF_023) 97Amino acid sequence of H7-SS-np_025 (based on H3 #265); (H7ssF_025) 98Amino acid sequence of H7-SS-np_026 (based on H3 #256); (H7ssF_026) 99Amino acid sequence of H7-SS-np_027 (based on H3 #249); (H7ssF_027) 100Amino acid sequence of H7-SS-np_028 (combine H7 #20 and #23); (H7ssF_028)101 Amino acid sequence of H7-SS-SA_01 (from H7-SS-np #16); (H7ssSA_01)102 Amino acid sequence of H7-SS-SA_02 (from H3-ss np #18); (H7ssSA_02)103Amino acid sequence of H10N8-SS-NP_01 (similar to H3 231, H7 16); (H10ssF_01)104Amino acid sequence of H10N8-SS-np_02 (similar to H3 262, H7 18); (H10ssF_02)105Amino acid sequence of H10N8-SS-np_03 (similar to H3 264, H7 20); (H10ssF_03)106Amino acid sequence of H10N8-SS-np_04 (similar to H3 256, H7 26); (H10ssF_04)107Amino acid sequence of H10N8-SS-np_05 (similar to H7 23); (H10ssF_05)108Amino acid sequence of H10N8-SS-np_06 (similar to H3 249, H7 27); (H10ssF_06)Protein Constructs With HA Joined to Transmembrane Domain 109Amino acid sequence of H3-SS-TM_231_HK68 110Amino acid sequence of H3-SS-TM_231_BK79 111Amino acid sequence of H3-SS TM_231_Wyo03 112Amino acid sequence of H3-SS-TM_231_Switz13 113Amino acid sequence of H3-SS-TM_256_Den05 114Amino acid sequence of H3-SS-TM_262_Den05 115Amino acid sequence of H3-SS-TM_264_Den05 116Amino acid sequence of H3-SS-TM_262_HK68 117Amino acid sequence of H3-SS-TM_262_BK79 118Amino acid sequence of H3-SS-TM_262_Wyo03 119Amino acid sequence of H3-SS-TM_262_Switz13 120Amino acid sequence of H7-SS-TM_016 121Amino acid sequence of H7-SS-TM_018 122Amino acid sequence of H7-SS-TM_020 123Amino acid sequence of H7-SS-TM_021 124Amino acid sequence of H7-SS-TM_023 125Amino acid sequence of H7-SS-TM_024 126Amino acid sequence of H7-SS-TM_025 127Amino acid sequence of H7-SS-TM_026 128Amino acid sequence of H7-SS_TM_027 (#16 with H7N7 A/England/268/1996)129Amino acid sequence of H7-SS_TM_028 (#16 with H7N7 A/Netherlands/219/2003)130 Amino acid sequence of H3-SS-TM_256_HK68 131Amino acid sequence of H3-SS-TM_258_HK68Protein Constructs With HA Joined to Monomeric Subunit 132Amino acid sequence of H3-SS-np_300 (based on 231 with glycan at N38 removed);(H3ssF_300) 133Amino acid sequence of H3-SS-np_301 (Delta cleavage loop; based on 231);(H3ssF_301) 134Amino acid sequence of H3-SS-np_302 (Delta cleavage loop; based on 258);(H3ssF_302) 135Amino acid sequence of H3-SS-np_303 (Delta cleavage loop; based on 231);(H3ssF_303) 136Amino acid sequence of H3-SS-np_304 (Delta cleavage loop; based on 231);(H3ssF_304) 137Amino acid sequence of H3-SS-np_305 (Delta cleavage loop; based on 231);(H3ssF_305) 138Amino acid sequence of H3-SS-np_306 (Glycan addition; based on 231); (H3ssF_306)139Amino acid sequence of H3-SS-np_307 (Glycan addition; based on 231); (H3ssF_307)140Amino acid sequence of H3-SS-np_308 (Glycan addition; based on 231); (H3ssF_308)141Amino acid sequence of H3-SS-np_309 (Glycan addition; based on 231); (H3ssF_309)142Amino acid sequence of H3-SS-np_310 (Glycan addition; based on 231); (H3ssF_310)143Amino acid sequence of H3-SS-np_311 (Glycan addition; based on 231); (H3ssF_311)144Amino acid sequence of H3-SS-np_312 (Glycan addition; based on 231); (H3ssF_312)145Amino acid sequence of H3-SS-np_313 (Glycan addition; based on 231); (H3ssF_313)146Amino acid sequence of H3-SS-np_314 (Glycan addition; based on 231); (H3ssF_314)147 Amino acid sequence of H3-SS-LS_08 (based on 249); (H3ssL_08) 148Amino acid sequence of H3-SS-LS_09 (based on 249 + 256); (H3ssL_09) 149Amino acid sequence of H3-SS-LS_10 (based on 249 + 258); (H3ssL_10) 150Amino acid sequence of H3-SS-LS_11 (based on 256); (H3ssL_11) 151Amino acid sequence of H3-SS-LS_12 (based on 258); (H3ssL_12) 152Amino acid sequence of H7-SS-LS_01 (based on H3 258); (H7ssL_01) 153Amino acid sequence of H7-SS-LS_02 (based on H3 249); (H7ssL_02) 154Amino acid sequence of H7-SS-LS_03 (based on H3 249 & 258); (H7ssL_03)155 Amino acid sequence of H7-SS-LS_04 (H7 20 + 26); (H7ssL_04) 156Amino acid sequence of H7-SS-LS_05 (H7 23 + 26); (H7ssL05) 157Amino acid sequence of H7-SS-LS_06 (H7 20 + 23 + 26); (H7ssL06) 158Amino acid sequence of H3-SS-np_256_HK68; (H3ssF_256) 159Amino acid sequence of H3-SS-np_258_HK68; (H3ssF_258)

The influenza viruses, and the sequences there from, listed above areexemplary, and any other Group 2 influenza virus, and sequences andproteins therefrom can be used to practice the invention.

The trimeric HA protein on the surface of the virus comprises a globularhead region and a stem, or stalk, region, which anchors the HA proteininto the viral lipid envelope. The head region of influenza HA is formedexclusively from a major portion of the HA1 polypeptide, whereas thestalk region is made from segments of HA1 and HA2. According to thepresent invention, the head region consists of the amino acids of aGroup 2 influenza HA protein corresponding to, approximately, aminoacids 60-329 of the full-length HA protein of influenza A virus(A/Denmark/35/2005 (H3N2)) (SEQ ID NO:4). Similarly, as used herein, thestem region is formed from the amino acids of a Group 2 influenza HAprotein corresponding to amino acids 1-59 and 330-519 of the full-lengthHA protein of influenza A virus (A/Denmark/35/2005 (H3N2)) (SEQ IDNO:4). As used herein, the term approximately, with regard to the headand stem regions means that the sequences cited above may vary in lengthby several (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids withoutaffecting the nature of the invention. Thus, for example, the headregion may consist of amino acids 64-329, amino acids 60-326 or aminoacids 62-327. Generally, the head and stem region will not vary from thelocations recited above by more than ten amino acids. In certain aspectsof the invention, the head region consists of the amino acid sequencebetween, and including, the amino acid residues corresponding to Cys68and Cys321 of influenza A virus (A/Denmark/35/2005 (H3N2)) (SEQ IDNO:4). With regard to HA proteins, it is understood by those skilled inthe art that HA proteins from different influenza viruses may havedifferent lengths due to sequence differences (insertions, deletions) inthe protein. Thus, reference to a corresponding region refers to aregion of another protein that is identical, or nearly so (e.g., atleast 90% identical, at least 95%, identical, at least 98% identical orat least 99% identical), in sequence, structure and/or function to theregion being compared. For example, with regard to the stem region of anHA protein, the corresponding region in another HA protein may not havethe same residue numbers, but will have a nearly identical sequence andwill perform the same function. As an example, in the embodiment statedabove, the head region of the HA protein from influenza virus A virus(A/Denmark/35/2005 (H3N2)) (SEQ ID NO:4) begins at amino acid 60. Thecorresponding amino acid at the beginning of the head region in A/NewCaledonia/20/1999 (H1) is amino acid C60. To better clarify sequencecomparisons between viruses, numbering systems are used by those in thefield, which relate amino acid positions to a reference sequence. Thus,corresponding amino acid residues in HA proteins from different strainsof influenza may not have the same residue number with respect to theirdistance from the n-terminal amino acid of the protein. For example,using the H3 numbering system, reference to residue 100 in A/NewCaledonia/20/1999 (1999 NC, H1) does not mean it is the 100^(th) residuefrom the N-terminal amino acid. Instead, residue 100 of A/NewCaledonia/20/1999 (1999 NC, H1) aligns with residue 100 of influenzaH3N2 strain. The use of such numbering systems is understood by thoseskilled in the art. While the H3 numbering system can be used toidentify the location of amino acids, unless otherwise noted, thelocation of amino acid residues in HA proteins will be identified bygeneral reference to the position of a corresponding amino acid from asequence disclosed herein.

The inventors have also discovered that by combining specific sequencesof the influenza virus HA protein with unrelated proteins, andnanoparticles made therefrom that are capable of presenting the HAprotein to the immune system, immune responses to targeted regions ofthe HA protein can be elicited. Thus, one embodiment of the presentinvention is a protein construct comprising a Group 2 influenza virus HAprotein joined to at least a portion of a monomeric subunit protein,wherein the head region of the Group 2 influenza virus HA protein hasbeen replaced with an amino acid sequence comprising less than 5contiguous amino acid residues from the head region of an influenza HAprotein, and wherein the protein construct is capable of forming ananoparticle.

By joining at least a portion of a Group 2 influenza HA protein to amonomeric subunit, protein constructs of the present invention arecapable of assembling into nanoparticles expressing trimers of Group 2influenza HA protein on their surface. Such trimers are in a pre-fusionform, and connection to the monomeric subunit, and expression on thenanoparticle stabilize the pre-fusion proteins in their trimeric form.Because of this, the HA protein is presented in a more native form,meaning certain surfaces of the stem polypeptides are not exposed,thereby reducing the risk that the stem polypeptides may induce anunfavorable antibody response.

In certain aspects, the at least a portion of a Group 2 influenza virusHA protein comprises at least one immunogenic portion from the stemregion of a Group 2 influenza virus HA protein, wherein the proteinconstruct elicits protective antibodies against an influenza virus. Incertain aspects, the at least a portion of a Group 2 influenza virus HAprotein comprises at least one immunogenic portion from the stem regionof an HA protein selected from the group consisting of an influenza H3virus HA protein, an influenza H4 virus HA protein, an H7 influenzavirus HA protein, an H10 influenza virus HA protein HA protein, an H14influenza virus HA protein, and an H15 influenza virus HA protein.

In certain aspects, the at least a portion of a Group 2 influenza virusHA protein comprises at least one immunogenic portion from the HAportion of a protein comprising an amino acid sequence at least 80%, atleast 85% identical, at least 90% identical, at least 95% identical, atleast 97% identical, or at least 99%, identical to a sequence selectedfrom the group consisting of SEQ ID NO:4-SEQ ID NO:26. In certainaspects, the at least a portion of a Group 2 influenza virus HA proteincomprises at least one immunogenic portion from the HA portion of aprotein comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:4-SEQ ID NO:26. In certain aspects, the at leasta portion of a Group 2 influenza virus HA protein comprises at least oneimmunogenic portion from the HA portion of a protein comprising an aminoacid sequence at least 80% identical, at least 85% identical, at least90% identical, at least 95% identical, at least 97% identical, or atleast 99% identical to a sequence selected from the group consisting ofSEQ ID NO: 47-SEQ ID NO:159. In certain aspects, the at least a portionof a Group 2 influenza virus HA protein comprises at least oneimmunogenic portion from the HA portion of a protein comprising an aminoacid sequence at least 80% identical, at least 85% identical, at least90% identical, at least 95% identical, at least 97% identical, or atleast 99%, identical to a sequence selected from the group consisting ofSEQ ID NO: 47-SEQ ID NO:159. In certain aspects, the at least a portionof a Group 2 influenza virus HA protein comprises at least oneimmunogenic portion from the HA portion of a protein comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:47-SEQ IDNO:159. In certain aspects, the at least a portion of a Group 2influenza virus HA protein comprises at least one immunogenic portionfrom the HA portion of a protein comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:47-SEQ ID NO:159. In oneembodiment protein constructs comprising immunogenic portions of a Group2 influenza HA protein elicit the production of broadly protectiveantibodies against influenza virus.

Immunogenic portions of proteins can comprise epitopes, which areclusters of amino acid residues that are recognized by the immunesystem, thereby eliciting an immune response. Such epitopes may consistof contiguous amino acids residues (i.e., amino acid residues that areadjacent to one another in the protein), or they may consist ofnon-contiguous amino acid residues (i.e., amino acid residues that arenot adjacent one another in the protein) but which are in close specialproximity in the finally folded protein. It is well understood by thoseskilled in the art that epitopes require a minimum of six amino acidresidues in order to be recognized by the immune system. Thus, incertain aspects the immunogenic portion from a Group 2 influenza HAprotein comprises at least one epitope. In one embodiment the at least aportion of a Group 2 influenza virus HA protein comprises at least 6amino acids, at least 10 amino acids, at least 25 amino acids, at least50 amino acids, at least 75 amino acids or at least 100 amino acids fromthe stem region of a Group 2 influenza HA protein. In certain aspectsthe at least a portion of a Group 2 influenza virus HA protein comprisesat least 6 amino acids, at least 10 amino acids, at least 25 aminoacids, at least 50 amino acids, at least 75 amino acids or at least 100amino acids from the stem region of a Group 2 influenza HA proteinselected from the group consisting of an influenza H3 virus HA protein,an influenza H4 virus HA protein, an H7 influenza virus HA protein, anH10 influenza virus HA protein HA protein, an H14 influenza virus HAprotein, and an H15 influenza virus HA protein. In certain aspects theat least a portion of a Group 2 influenza virus HA protein comprises atleast 6 amino acids, at least 10 amino acids, at least 25 amino acids,at least 50 amino acids, at least 75 amino acids or at least 100 aminoacids from the stem region of a Group 2 influenza HA protein having anamino acid sequence at least 80% identical, at least 85% identical, atleast 90% identical, at least 95% identical, at least 97% identical, orat least 99% identical to an HA protein from an influenza virus selectedfrom those listed in Table 2. In certain aspects the at least a portionof a Group 2 influenza virus HA protein comprises at least 6 aminoacids, at least 10 amino acids, at least 25 amino acids, at least 50amino acids, at least 75 amino acids or at least 100 amino acids fromthe stem region of a Group 2 influenza HA protein from an influenzavirus selected from those listed in Table 2, and variants thereof. Incertain aspects the at least a portion of a Group 2 influenza virus HAprotein comprises at least 6 amino acids, at least 10 amino acids, atleast 25 amino acids, at least 50 amino acids, at least 75 amino acidsor at least 100 amino acids from the stem region of a Group 2 influenzaHA protein comprising a sequence at least 80%, at least 85% identical,at least 90% identical, at least 95% identical, at least 97% identical,or at least 99%, identical to a sequence selected from the groupconsisting of SEQ ID NO:4-SEQ ID NO:26. In certain aspects the at leasta portion of a Group 2 influenza virus HA protein comprises at least 6amino acids, at least 10 amino acids, at least 25 amino acids, at least50 amino acids, at least 75 amino acids or at least 100 amino acids fromthe stem region of a Group 2 influenza HA protein comprising a sequenceselected from the group consisting of SEQ ID NO:4-SEQ ID NO:26. Incertain aspects the at least a portion of a Group 2 influenza virus HAprotein comprises at least 6 amino acids, at least 10 amino acids, atleast 25 amino acids, at least 50 amino acids, at least 75 amino acidsor at least 100 amino acids from the HA portion of a protein comprisinga sequence at least 80%, at least 85% identical, at least 90% identical,at least 95% identical, at least 97% identical, or at least 99%,identical to a sequence selected from the group consisting of SEQ IDNO:47-SEQ ID NO:159. In certain aspects the at least a portion of aGroup 2 influenza virus HA protein comprises at least 6 amino acids, atleast 10 amino acids, at least 25 amino acids, at least 50 amino acids,at least 75 amino acids or at least 100 amino acids from the HA portionof a protein comprising a sequence selected from the group consisting ofSEQ ID NO:47-SEQ ID NO:159.

In certain aspects of the invention, the amino acids are contiguousamino acids from the stem region of a Group 2 influenza virus HAprotein. In certain aspects, protein constructs of the inventioncomprising at least 6 amino acids, at least 10 amino acids, at least 25amino acids, at least 50 amino acids, at least 75 amino acids or atleast 100 amino acids from the stem region of a Group 2 influenza virusHA protein elicit the production of broadly protective antibodiesagainst influenza virus. In certain aspects of the invention, a proteinconstruct comprises at least 6 amino acids, at least 10 amino acids, atleast 25 amino acids, at least 50 amino acids, at least 75 amino acidsor at least 100 amino acids from the stem region of a Group 2 influenzavirus HA protein comprising an amino acid sequence at least 80%identical, at least 85% identical, at least 90% identical, at least 95%identical, at least 97% identical, or at least 99% identical to sequenceselected from the group consisting of SEQ ID NO:4-SEQ ID NO:26. Incertain aspects of the present invention, a protein construct comprisesat least 6 amino acids, at least 10 amino acids, at least 25 aminoacids, at least 50 amino acids, at least 75 amino acids or at least 100amino acids from the stem region of a Group 2 influenza virus HA proteincomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:4-SEQ ID NO:26. In certain aspects, the amino acids arenon-contiguous, but are in close spatial proximity in the final protein.

While the present application exemplifies the use of stem regionsequences from several exemplary Group 2 influenza virus HA proteins,the invention may also be practiced using stem regions from proteinscomprising variations of the disclosed Group 2 influenza HA sequences.Thus, in certain aspects of the invention, the Group 2 influenza HAprotein is from a virus selected from the Group 2 viruses listed inTable 2, and variants thereof. In certain aspects, the Group 2 influenzavirus HA protein comprises an amino acid sequence at least 80%, at least85%, at least 90%, at least 92%, at least 94%, at least 96%, at least98% or at least 99% identical the stem region of a Group 2 influenzavirus HA protein comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO:4-SEQ ID NO:26. In certain aspects, theGroup 2 influenza HA protein comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NO:4-SEQ ID NO:26.

In certain aspects of the invention, the head region sequence of the HAprotein in the protein construct is replaced with a linker sequence. Anylinker sequence may be used so long as the stem region sequences areable to adopt the desired conformation. While any amino acids may beused to make the linker sequence, in certain aspects of the inventionthe amino acids lack large or charged side chains. Preferred amino acidsto use include, but are not limited to, cysteine, serine, glycine,alanine, valine and proline. In one embodiment, the linker is made fromone or more amino acids selected from the group consisting of serine,glycine, cysteine, valine, proline and/or phenylalanine residues. Incertain embodiments, it may be desirable to include an amino acidresidue, the side chain of which is capable of forming a covalent bond,such as a disulfide bond, with another amino acid. One example of suchan amino acid is cysteine. The length of the linker sequence may vary,but preferred embodiments use the shortest possible sequence in order toallow the stem sequences to form the desired structure. In certainaspects, the linker sequence is less than 12 amino acids in length. Inone embodiment, the linker sequence is less than 10 amino acids inlength. In one embodiment, the linker sequence is less than 5 aminoacids in length. In preferred embodiments, the linker sequence lackscontiguous amino acid sequences from the head region of an HA protein.In certain aspects, the linker sequence comprises less than 5 contiguousamino acids from the head region of an HA protein. In certain aspectsthe head region sequence is replaced with an amino acid sequencecomprising SEQ ID N034, SEQ ID NO:35, or variants thereof.

The inventors have also discovered that the stability of proteinconstructs and nanoparticles of the invention can be improved by makingfurther alterations to the Group 2 influenza virus HA protein of thedisclosed protein constructs. For example, the inventors have discoveredthat extending the length of helix A improves the performance of proteinconstructs of the invention. Thus, one embodiment is a protein constructof the invention in which helix A has been extended by the addition ofamino acids. One embodiment is a protein construct of the invention,wherein the protein construct comprises a Group 2 influenza virus HAprotein joined to at least a portion of a monomeric subunit, wherein thehead region of the Group 2 influenza virus HA protein has been replacedwith an amino acid sequence comprising less than 5 contiguous amino acidresidues from the head region of an influenza HA protein, and whereinthe carboxy-terminal end of helix A (i.e., the portion that links to theamino end of helix C) has been extended by the addition of amino acidresidues. It should be appreciated that because the goal is to extendthe helix, the sequence of amino acids added to the carboxy-terminal endof helix A should preferably form a helix. In certain aspects of theinvention, the length of helix A is extended by adding an amino acidsequence comprising SEQ ID NOs:36 or 37, or helix-forming variantsthereof, to the carboxyl-end of helix A. In certain aspects of theinvention, the length of helix A is extended by adding a sequencecomprising, or consisting of, X₁LMX₂Q (SEQ ID NO: 160), or helix-formingvariants thereof, to the carboxyl-end of helix A, wherein the aminoacids at positions X₁ and X₂ are acidic amino acids. It should be notedthat X₁ and X₂ can, but need not, be the same amino acid residue. Incertain aspects, the residues at the first and fourth position of such alinker are selected from the group consisting of glutamine, glutamicacid, asparagine, aspartic acid, glycine, and proline. In oneembodiment, helix A is extended by adding an amino acid sequenceconsisting of SEQ ID NOs:36 or 37, or helix-forming variants thereof, tothe carboxyl-end of helix A. In certain aspects of the invention, thelength of helix A is extended by adding a sequence comprising ALMAQ (SEQID NO: 36) or ELMEQ (SEQ ID NO: 37), or helix-forming variants thereof,to the carboxyl-end of helix A. In certain aspects of the invention, thelength of helix A is extended by adding a sequence consisting of ALMAQ(SEQ ID NO: 36) or ELMEQ (SEQ ID NO: 37), or helix-forming variantsthereof, to the carboxyl-end of helix A.

In addition to extension of helix A, the inventors have discovered thatmodification of the amino acid sequence joining the carboxyl-end ofhelix A to the amino-end of helix C (herein referred to as theinter-helix region or inter-helix loop, one example of which isrepresented by SEQ ID NO:38), improves the stability and performance ofprotein constructs and nanoparticles of the invention. Moreparticularly, the inventors have found that shortening the length of theinter-helix region improves the stability and performance of proteinconstructs and nanoparticles of the invention. Thus, in certain aspectsof the invention, the amino acid sequence joining the carboxyl-end ofhelix A to the amino-end of helix C in a protein construct of theinvention is modified to improve the stability of a protein construct ofthe invention. It should be appreciated that improving the stability ofa protein construct of the invention means stabilizing thethree-dimensional structure of a protein construct of the invention, andin particular the stem-region of a protein construct of the invention,such that it approximates the three-dimensional structure of the stemregion of a native Group 2 influenza HA protein, and is able to elicitan immune response to a Group 2 influenza virus. Thus, in certainaspects of the invention, the inter-helix region of a protein constructof the invention is shortened. Such shortening can be achieved byremoving amino acids from the existing inter-helix region, or byreplacing amino acids of the inter-helix region with a linker sequence.In certain aspects, the inter-helix region of a protein construct of theinvention is shortened to less than 6 amino acids. In certain aspects,amino acids of the inter-helix region are replaced with a linkersequence. In certain aspects of the invention, amino acids of aninter-helix region corresponding to the inter-helix region of aninfluenza virus A (Denmark/35/2005(H3N2)) HA protein (SEQ ID NO:4) arereplaced with a linker sequence. In certain aspects of the invention,amino acids of an inter-helix region corresponding to amino acids402-437 of an influenza virus A (Denmark/35/2005(H3N2)) HA protein (SEQID NO:4) are replaced with a linker sequence. In certain aspects of theinvention, an inter-helix region comprising amino acids 402-437 of SEQID NO:4 is replaced with a linker sequence. In certain aspects of theinvention, an inter-helix region corresponding to a region of influenzavirus A (Denmark/35/2005(H3N2)) HA protein (SEQ ID NO:4) represented bySEQ ID NO:38 is replaced with a linker sequence. In certain aspects ofthe invention, an inter-helix region of the Group 2 influenza virus HAprotein comprising an amino acid sequence at least 90%, at least 97%, atleast 99% identical to SEQ ID NO: 38, is replaced with a linkersequence. In one embodiment, a region of the Group 2 influenza virus HAprotein comprising SEQ ID NO: 38, is replaced with a linker sequence. Incertain aspects of the invention, a region of the Group 2 influenzavirus HA protein consisting of SEQ ID NO: 38, is replaced with a linkersequence. In certain aspects of the invention, the inter-helix region isreplaced with a linker sequence comprising GGPD (SEQ ID NO:39). Incertain aspects of the invention, an inter-helix region corresponding toamino acids 402-437 of SEQ ID NO:4 is replaced with a linker sequencehaving the physical spatial, and/or chemical properties of a peptideconsisting of GGPD (SEQ ID NO:39). In certain aspects of the invention,an inter-helix region corresponding to amino acids 402-437 of SEQ IDNO:4 is replaced with a linker sequence having the propensity to form ahelix. In certain aspects of the invention, an inter-helix regioncorresponding to amino acids 402-437 of SEQ ID NO:4 is replaced with alinker sequence comprising GGPD (SEQ ID NO:39), or conservative variantsthereof. In certain aspects of the invention, the inter-helix region isreplaced with a linker sequence consisting of GGPD (SEQ ID NO:39).

As has been previously described, protein constructs of the inventioncan contain one, several or all of the mutations and sequencealterations described herein. Thus, for example, a protein construct inwhich helix A has been extended, as described supra, can also have theinter-helix region shortened or replaced with a linker sequence, asdescribed supra. Thus, one aspect of the invention is a proteinconstruct comprising a Group 2 influenza virus HA protein joined to atleast a portion of a monomeric subunit protein, wherein the head regionof the Group 2 influenza virus HA protein has been replaced with anamino acid sequence comprising less than 5 contiguous amino acidresidues from the head region of an influenza HA protein, wherein theinter-helix region has been shortened or replaced with a linkersequence, and wherein the protein construct is capable of forming ananoparticle. Methods of replacing the HA protein head region, andmethods of shortening or replacing the inter-helix region are disclosedherein. It should be understood that in embodiments in which thecarboxyl end of helix A has been extended by the addition of aminoacids, the inter-helix region would be replaced with a linker that joinsthe amino-terminal end of helix C with the carboxyl-terminal end of theextension sequence of helix A.

The inventors have further discovered that the stability of proteinconstructs of the invention can be improved by making site-specificmutations in the sequence of the Group 2 influenza virus stem region. Inparticular, mutations that form ionic bonds, salt bridges, of thatincrease hydrophobic packing, and the like, can strengthen the stabilityof protein constructs and nanoparticles of the invention. Thus, incertain aspects of the invention, a protein construct of the inventioncomprises one or more mutations that forms or strengthens an ionicinteraction, or a salt bridge, or that increases hydrophobic packing.Any type of mutation that has the desired effect of increasing thestability of a protein construct of the invention can be made, althoughsubstitution mutations are preferred. In certain aspects of theinvention, a mutation is made in the Group 2 influenza virus HA proteinat an amino acid location corresponding to a location in SEQ ID NO:4selected from the group consisting of K396, L397, L400, S438, N440,E448, T452 and N461. In one embodiment, the amino acid corresponding toK396 in the influenza virus A (Denmark/35/2005(H3N2) HA protein (SEQ IDNO:4) is changed to an amino acid residue selected from the groupconsisting of methionine, leucine, isoleucine, alanine and valine. Incertain aspects of the invention, the amino acid corresponding to K396in the influenza virus A (Denmark/35/2005(H3N2) HA protein (SEQ ID NO:4)is changed to a methionine or a leucine. In one embodiment, the aminoacid corresponding to L397 in the influenza virus A(Denmark/35/2005(H3N2) HA protein (SEQ ID NO:4) is changed to an aminoacid residue selected from the group consisting of methionine, leucine,isoleucine, alanine and valine. In certain aspects of the invention, theamino acid corresponding to L397 in the influenza virus A(Denmark/35/2005(H3N2) HA protein (SEQ ID NO:4) is changed to a valine.In certain aspects of the invention, the amino acid corresponding toL400 in the influenza virus A (Denmark/35/2005(H3N2) HA protein (SEQ IDNO:4) is changed to an amino acid residue selected from the groupconsisting of methionine, leucine, isoleucine, alanine and valine. Incertain aspects of the invention, the amino acid corresponding to L400in the influenza virus A (Denmark/35/2005(H3N2) HA protein (SEQ ID NO:4)is changed to a valine. In certain aspects of the invention, the aminoacid corresponding to S438 in the influenza virus A(Denmark/35/2005(H3N2) HA protein (SEQ ID NO:4) is changed to an aminoacid residue selected from the group consisting of asparagine,glutamine, serine, threonine, and cysteine. In certain aspects of theinvention, the amino acid corresponding to S438 in the influenza virus A(Denmark/35/2005(H3N2) HA protein (SEQ ID NO:4) is changed to acysteine. In certain aspects of the invention, the amino acidcorresponding to N440 in the influenza virus A (Denmark/35/2005(H3N2) HAprotein (SEQ ID NO:4) is changed to an amino acid residue selected fromthe group consisting of methionine, leucine, isoleucine, alanine andvaline. In certain aspects of the invention, the amino acidcorresponding to N440 in the influenza virus A (Denmark/35/2005(H3N2) HAprotein (SEQ ID NO:4) is changed to a leucine. In certain aspects of theinvention, the amino acid corresponding to E448 in the influenza virus A(Denmark/35/2005(H3N2) HA protein (SEQ ID NO:4) is changed to an aminoacid residue selected from the group consisting of methionine, leucine,isoleucine, alanine and valine. In certain aspects of the invention, theamino acid corresponding to E448 in the influenza virus A(Denmark/35/2005(H3N2) HA protein (SEQ ID NO:4) is changed to a leucine.In certain aspects of the invention, the amino acid corresponding toT452 in the influenza virus A (Denmark/35/2005(H3N2) HA protein (SEQ IDNO:4) is changed to an amino acid residue selected from the groupconsisting of methionine, leucine, isoleucine, alanine and valine. Incertain aspects of the invention, the amino acid corresponding to T452in the influenza virus A (Denmark/35/2005(H3N2) HA protein (SEQ ID NO:4)is changed to a valine. In certain aspects of the invention, the aminoacid corresponding to N461 in the influenza virus A(Denmark/35/2005(H3N2) HA protein (SEQ ID NO:4) is changed to an aminoacid residue selected from the group consisting of histidine, lysine,glutamic acid, aspartic acid, and arginine. In certain aspects of theinvention, the amino acid corresponding to N461 in the influenza virus A(Denmark/35/2005(H3N2) HA protein (SEQ ID NO:4) is changed to an aminoacid residue selected from the group consisting of histidine, lysine,and arginine. In certain aspects of the invention, the amino acidcorresponding to N461 in the influenza virus A (Denmark/35/2005(H3N2) HAprotein (SEQ ID NO:4) is changed to an arginine.

Additional mutations that may stabilize protein constructs of theinvention include a mutation at an amino acid location corresponding toa location in SEQ ID NO:4 selected from the group consisting of G39,T46, N54, T58, L331, N338, and Q392. It should be understood thatmutations at such locations can include those in which the amino acidbeing inserted is similar in properties to those suggested herein.

In certain aspects of the invention, the amino acid corresponding to G39in the influenza virus A (Denmark/35/2005(H3N2) HA protein (SEQ ID NO:4)is changed to an amino acid residue selected from the group consistingof cysteine, serine, threonine, proline, asparagine, and glutamine. Incertain aspects of the invention, the amino acid corresponding to G39 inthe influenza virus A (Denmark/35/2005(H3N2) HA protein (SEQ ID NO:4) ischanged to a cysteine.

In certain aspects of the invention, the amino acid corresponding to T46in the influenza virus A (Denmark/35/2005(H3N2) HA protein (SEQ ID NO:4)is changed to an amino acid residue selected from the group consistingof cysteine, serine, threonine, proline, asparagine, and glutamine. Incertain aspects of the invention, the amino acid corresponding to T46 inthe influenza virus A (Denmark/35/2005(H3N2) HA protein (SEQ ID NO:4) ischanged to a cysteine.

In certain aspects of the invention, the amino acid corresponding to N54in the influenza virus A (Denmark/35/2005(H3N2) HA protein (SEQ ID NO:4)is changed to an amino acid residue selected from the group consistingof histidine, arginine and lysine. In certain aspects of the invention,the amino acid corresponding to N54 in the influenza virus A(Denmark/35/2005(H3N2) HA protein (SEQ ID NO:4) is changed to ahistidine.

In certain aspects of the invention, the amino acid corresponding to T58in the influenza virus A (Denmark/35/2005(H3N2) HA protein (SEQ ID NO:4)is changed to an amino acid residue selected from the group consistingof methionine, leucine, isoleucine, alanine and valine. In certainaspects of the invention, the amino acid corresponding to T58 in theinfluenza virus A (Denmark/35/2005(H3N2) HA protein (SEQ ID NO:4) ischanged to a leucine.

In certain aspects of the invention, the amino acid corresponding toL331 in the influenza virus A (Denmark/35/2005(H3N2) HA protein (SEQ IDNO:4) is changed to an amino acid residue selected from the groupconsisting of histidine, arginine and lysine. In certain aspects of theinvention, the amino acid corresponding to L331 in the influenza virus A(Denmark/35/2005(H3N2) HA protein (SEQ ID NO:4) is changed to a lysine.

In certain aspects of the invention, the amino acid corresponding toN338 in the influenza virus A (Denmark/35/2005(H3N2) HA protein (SEQ IDNO:4) is changed to an amino acid residue selected from the groupconsisting of cysteine, serine, proline, asparagine, glutamine, andthreonine. In certain aspects of the invention, the amino acidcorresponding to N338 in the influenza virus A (Denmark/35/2005(H3N2) HAprotein (SEQ ID NO:4) is changed to a cysteine.

In certain aspects of the invention, the amino acid corresponding toQ392 in the influenza virus A (Denmark/35/2005(H3N2) HA protein (SEQ IDNO:4) is changed to an amino acid residue selected from the groupconsisting of cysteine, serine, proline, asparagine, glutamine, andthreonine. In certain aspects of the invention, the amino acidcorresponding to Q392 in the influenza virus A (Denmark/35/2005(H3N2) HAprotein (SEQ ID NO:4) is changed to a cysteine.

In addition to the above, the inventors have discovered that mutationsadding glycan linkage sites can be beneficial. Thus, in certain aspectsof the invention, the protein construct comprise one or more mutations,or one or more pairs of mutations, selected from the group consisting ofQ49N/E51T (mutation to add a group 1 glycan), E56N/V59T (mutations inhead linker and adjacent residue), V59N/P61T (mutations in head linker),G62N/G64T (mutations in head linker), V329N/L331T (mutations in headlinker and adjacent residue), L331N/L333T, D437N/Y439T (mutations ininterhelix linker and adjacent residue), Q432N/G434T (inserted G)(mutations in interhelix linker and adjacent residue), Q372N/S374T, andA492N/I494T.

In addition, in certain aspects of the invention, the loop correspondingto amino acids 339-357 in the influenza virus A (Denmark/35/2005(H3N2)HA protein (SEQ ID NO:4) can be replaced with a glycine linker.

As has been previously described, protein constructs of the inventioncan contain one, several or all of the mutations and sequencealterations described herein. Thus, for example, a protein construct inwhich helix A has been extended, as described herein, can also have theinter-helix region shortened or replaced with a linker sequence, asdescribed herein, and can also contain one or more of the site-specificmutations described herein. Thus, one aspect of the invention is aprotein construct comprising a Group 2 influenza virus HA protein joinedto at least a portion of a monomeric subunit protein, wherein the headregion of the Group 2 influenza virus HA protein has been replaced withan amino acid sequence comprising less than 5 contiguous amino acidresidues from the head region of an influenza HA protein, wherein theinter-helix region has been shortened or replaced with a linkersequence, wherein the HA portion of the protein construct comprises oneor more site-specific mutation at a location corresponding to a locationin SEQ ID NO:4 selected from the group consisting of K396, L397, L400,S438, N440, E448, T452, N461, G39, T46, N54, T58, L331, N338, and D437,and wherein the protein construct is capable of forming a nanoparticle.Such constructs may also comprise one or more mutations, or one or morepairs of mutations, selected from the group consisting of Q49N/E51T,E56N/V59T (mutations in head linker and adjacent residue), V59N/P61T(mutations in head linker), G62N/G64T (mutations in head linker),V329N/L331T (mutations in head linker and adjacent residue),L331N/L333T, D437N/Y439T (mutations in interhelix linker and adjacentresidue), Q432N/G434T (inserted G) (mutations in interhelix linker andadjacent residue), Q372N/S374T, and A492N/I494T. Methods of replacingthe HA protein head region, extending helix A, shortening or replacingthe inter-helix region, and suitable site-specific mutations have beendisclosed herein. It should be understood that in embodiments in whichthe carboxyl end of helix A has been extended by the addition of aminoacids, the inter-helix region would be replaced with a linker that joinsthe amino-terminal end of helix C with the carboxyl-terminal end of theextension sequence of helix A.

Heretofore has been described specific aspects of a protein construct ofthe invention, useful for producing nanoparticle vaccines. To aid inclarifying the invention, the inventors will now describe variousaspects in alternative and greater detail. It should be understood thatany aspects of the invention described below also apply to embodimentsand aspects of protein constructs already described herein.

Protein constructs of the present invention can be made usingrecombinant technology to link together various portions of Group 3influenza HA proteins, and make sequences alterations thereto.Recombinant technology can also be used to add appropriate linkers andmonomeric subunits. In this way, protein constructs can be produced thatcomprise specific sequences necessary to produce protein constructs andconsequently, nanoparticle vaccines of the invention. Thus, oneembodiment of the present invention is a protein construct (alsoreferred to herein as a fusion protein) comprising a first amino acidsequence from the stem region of a Group 2 influenza virus HA proteinand a second amino acid sequence from the stem region of a Group 2influenza virus HA protein, the first and second amino acid sequencesbeing covalently linked by a linker sequence,

-   -   wherein the first amino acid sequence comprises at least 20        contiguous amino acid residues from the amino acid sequence        upstream of the amino-terminal end of the head region sequence;    -   wherein the second amino acid sequence comprises at least 20        contiguous amino acid residues from the amino acid sequence        downstream of the carboxyl-terminal end of the head region        sequence; and,    -   wherein the first or second amino acid sequence is joined to at        least a portion of a monomeric subunit domain such that the        protein construct is capable of forming a nanoparticle.

In certain aspects of the invention, the first amino acid sequence isfrom the stem region of a Group 2 influenza virus HA protein from avirus selected from the group consisting of an influenza H3 virus HAprotein, an influenza H4 virus HA protein, an H7 influenza virus HAprotein, an H10 influenza virus HA protein HA protein, an H14 influenzavirus HA protein, and an H15 influenza virus HA protein. In certainaspects of the invention, the first amino acid sequence is from the stemregion of an HA protein from a Group 2 virus listed in Table 2. Incertain aspects of the invention, the first amino acid sequence is fromthe stem region of a Group 2 influenza HA protein, wherein the HAprotein comprises an amino acid sequences at least 85%, at least 90%, atleast 95% or at least 97% identical to a sequence selected from thegroup consisting of SEQ ID NO:4-SEQ ID NO:26 and SEQ ID NO:47-SEQ IDNO:159. In certain aspects of the invention, the first amino acidsequence is from the stem region of a Group 2 influenza HA protein,wherein the HA protein comprises a sequence selected from the groupconsisting of SEQ ID NO:4-SEQ ID NO:26 and SEQ ID NO:47-SEQ ID NO:159.

In certain aspects of the invention, the second amino acid sequence isfrom the stem region of a Group 2 influenza HA protein from a virusselected from the group consisting of an influenza H3 virus, aninfluenza H4 virus, an H7 influenza virus, an H10 influenza virus, anH14 influenza virus, and an H15 influenza virus. In certain aspects ofthe invention, the second amino acid sequence is from the stem region ofan HA protein from a Group 2 virus listed in Table 2. In certain aspectsof the invention, the second amino acid sequence is from the stem regionof a Group 2 influenza virus HA protein, wherein the HA proteincomprises an amino acid sequences at least 85%, at least 90%, at least95% or at least 97% identical to a sequence selected from the groupconsisting of SEQ ID NO:4-SEQ ID NO:26 and SEQ ID NO:47-SEQ ID NO:159.In certain aspects of the invention, the second amino acid sequence isfrom the stem region of a Group 2 influenza virus HA protein comprisinga sequence selected from the group consisting of SEQ ID NO:4-SEQ IDNO:26 and SEQ ID NO:47-SEQ ID NO:159.

As noted above, the first amino acid sequence comprises at least 20contiguous amino acid residues from the amino acid sequence upstream ofthe amino-terminal end of the head region sequence. According to thepresent invention, the term upstream refers to the entirety of the aminoacid sequence linked to the amino-terminal end of the first amino acidresidue of the head region. Preferred upstream sequences are those thatare immediately adjacent to the head region sequence. In certain aspectsof the invention, the amino-terminal end of the head region is locatedat the amino acid residue corresponding to Q60 of the HA protein ofinfluenza A (Denmark/35/2005 (H3N2)) HA protein (SEQ ID NO:4) In certainaspects of the invention, the first amino acid sequence comprises atleast 20 contiguous amino acid residues from the region of a Group 2influenza virus HA protein corresponding to amino acid residues 1-59 ofthe HA protein of influenza A Denmark/35/2005 (H3N2)) represented by SEQID NO:4. In certain aspects of the invention, the first amino acidsequence comprises at least 20 contiguous amino acid residues from asequence at least 85%, at least 90%, at least 95% or at least 97%identical to a sequence selected from the group consisting of SEQ IDNO:27, SEQ ID NO:28 and SEQ ID NO:29. In certain aspects of theinvention, the first amino acid sequence comprises at least 20contiguous amino acid residues from a sequence selected from the groupconsisting of SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29.

In certain aspects of the invention, the first amino acid sequencecomprises at least 40 contiguous amino acid residues from the amino acidregion of an HA protein corresponding to amino acid residues 1-59 ofinfluenza A Denmark/35/2005 (H3N2)) HA protein (SEQ ID NO:4). In certainaspects of the invention, the first amino acid sequence comprises atleast 40 contiguous amino acid residues from a sequence at least 85%, atleast 90%, at least 95% or at least 97% identical to SEQ ID NO:27 or SEQID NO:28. In certain aspects of the invention, the first amino acidsequence comprises at least 40 contiguous amino acid residues from SEQID NO:27 or SEQ ID NO:28.

In certain aspects of the invention, the first amino acid sequencecomprises a sequence at least 85%, at least 90%, at least 95% or atleast 97% identical to SEQ ID NO:27. In one embodiment, the first aminoacid sequence comprises SEQ ID NO:27.

As noted above, the second amino acid sequence comprises at least 20contiguous amino acid residues from the amino acid sequence downstreamof the carboxyl-terminal end of the head region sequence. According tothe present invention, the term downstream refers to the entirety of theamino acid sequence linked to the carboxyl-terminal amino acid residueof the head region. Preferred upstream sequences are those that areimmediately adjacent to the head region sequence. In certain aspects ofthe invention, the carboxyl-terminal end of the head region is locatedat the amino acid position corresponding to T329 of the HA protein ofinfluenza A (Denmark/35/2005(H3N2)) HA protein represented by SEQ IDNO:4. Thus, in certain aspects of the invention, the second amino acidsequence comprises at least 20 contiguous amino acids from a region of aGroup 2 influenza HA protein corresponding to amino acid residues330-519 of influenza A (Denmark/35/2005) (H3N2) HA protein. In certainaspects of the invention, the second amino acid sequence comprises atleast 20 contiguous amino acids from a region of a Group 2 influenza HAprotein comprising amino acid residues 330-519 of influenza A(Denmark/35/2005(H3N2)) (SEQ ID NO:4). In one embodiment, the secondamino acid sequence comprises at least 20 contiguous amino acid residuesfrom a sequence at least 85%, at least 90%, at least 95% or at least 97%identical to a sequence selected from the group consisting of SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33. In one embodiment,the second amino acid sequence comprises at least 20 contiguous aminoacid residues from a sequence selected from the group consisting of SEQID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33.

In certain aspects of the invention, the second amino acid sequencecomprises at least 40 contiguous amino acids from a region of a Group 2influenza HA protein corresponding to amino acid residues 330-519 ofinfluenza A (Denmark/35/2005) (H3N2) HA protein. In certain aspects ofthe invention, the second amino acid sequence comprises at least 40contiguous amino acids from a region of a Group 2 influenza HA proteincomprising amino acid residues 330-519 of influenza A(Denmark/35/2005(H3N2)) (SEQ ID NO:4). In certain aspects of theinvention, the second amino acid sequence comprises at least 40contiguous amino acid residues from a sequence at least 85%, at least90%, at least 95% or at least 97% identical to a sequence selected fromthe group consisting of SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32. Incertain aspects of the invention, the second amino acid sequencecomprises at least 20 contiguous amino acid residues from a sequenceselected from the group consisting of SEQ ID NO:30, SEQ ID NO:31, andSEQ ID NO:32.

In certain aspects of the invention, the second amino acid sequencecomprises an amino acid sequence at least 85%, at least 90%, at least95% or at least 97% identical to SEQ ID NO:36. In one embodiment, thesecond amino acid sequence comprises SEQ ID NO:36.

In certain aspects of the invention, the second amino acid sequencecomprises at least 60, at least 72, at least 75, at least 100, at least150, at least 175, or at least 190 contiguous amino acids from a regionof a Group 2 influenza HA protein corresponding to amino acid residues330-519 of influenza A (Denmark/35/2005) (H3N2) HA protein. In certainaspects of the invention, the second amino acid sequence comprises atleast 60, at least 72, at least 75, at least 100, at least 150, at least175, or at least 190 contiguous amino acids from a region of a Group 2influenza HA protein comprising amino acid residues 330-519 of influenzaA (Denmark/35/2005(H3N2)) (SEQ ID NO:4). In certain aspects of theinvention, the second amino acid sequence comprises at least 40, atleast 60, at least 72, at least 75, at least 100, at least 150, at least175, or at least 190 contiguous amino acid residues from a sequence atleast 85%, at least 90%, at least 95% or at least 97% identical to SEQID NO:30. In one embodiment, the second amino acid sequence comprises atleast 40, at least 60, at least 72, at least 75, at least 100, at least150, at least 175, or at least 190 contiguous amino acid residues fromSEQ ID NO:30.

As noted above, the first and second amino acid sequences of the proteinconstruct can be joined by a linker sequence. Any linker sequence can beused as long as the linker sequence has less than five contiguous aminoacid residues from the head region of an HA protein and so long as thefirst and second amino acids are able to form the desired conformation.In one embodiment, the linker sequence is less than 10 amino acids, lessthan 7 amino acids or less than 5 amino acids in length. In oneembodiment, the linker sequence comprises glycine and serine. In oneembodiment, the linker sequence joins the carboxyl-terminal end of thefirst amino acid sequence to the amino-terminal end of the second aminoacid sequence. In certain aspects of the invention, the linker sequencejoins the carboxyl-terminal end of the second amino acid sequence to theamino-terminal end of the first amino acid sequence. In certain aspectsof the invention, the linker sequence is similar in chemical and specialproperties to a peptide consisting of SEQ ID NO:34 or SEQ ID NO:35. Incertain aspects of the invention, the linker comprises SEQ ID NO:34 orSEQ ID NO:35, or conservative variants thereof. In one embodiment, thelinker comprises SEQ ID NO:34 or SEQ ID NO:35. In certain aspects of theinvention, the linker consists of SEQ ID NO:34 or SEQ ID NO:35.

In certain aspects of the invention, the second amino acid sequencecomprises an amino acid sequence from a Group 2 influenza virus HAprotein, corresponding to amino acids 330-519 of influenza A(Denmark/35/2005 (H3N2)) HA protein (SEQ ID NO:4), wherein the regioncorresponding to the inter-helix region of the HA protein (SEQ ID NO:4)is replaced with a linker peptide. In certain aspects of the invention,the inter-helix region of the influenza A (Denmark/35/2005 (H3N2)) HAprotein (SEQ ID NO:4) consists essentially of amino acids 402-437 of SEQID NO:4. Thus, in certain aspects of the invention, the second aminoacid sequence comprises an amino acid sequence from a Group 2 influenzavirus HA protein, corresponding to amino acids 330-519 of influenza A(Denmark/35/2005 (H3N2)) HA protein (SEQ ID NO:4), wherein the regioncorresponding to amino acids 402-437 of SEQ ID NO:4 is replaced with alinker peptide. In certain aspects of the invention, the second aminoacid sequence comprises an amino acid sequence at least 80% identical,at least 85% identical, at least 90% identical, at least 95% identical,at least 97% identical or at least 99% identical to SEQ ID NO:30,wherein the region corresponding to the inter-helix region (i.e., aminoacids 402-437 of SEQ ID NO:4), is replaced with a linker peptide. Incertain aspects of the invention, the second amino acid sequencecomprises SEQ ID NO:30, wherein the region corresponding to theinter-helix region (i.e., amino acids 402-437 of SEQ ID NO:4), isreplaced with a linker peptide. In certain aspects of the invention, thesecond amino acid sequence comprises SEQ ID NO:30, wherein amino acids73-108 of SEQ ID NO:30 are replaced with a linker peptide. Any linkersequence can be used as the linker peptide in the second amino acidsequence, as long as the protein construct is able to form the desiredconformation. In certain aspects of the invention, the linker peptide isless than 10 amino acids, less than 7 amino acids or less than 5 aminoacids in length. In one embodiment, the linker peptide is four aminoacids in length. In certain aspects of the invention, the linkersequence comprises one or more amino acids selected from the groupconsisting of glycine, serine, proline and aspartic acid. In certainaspects of the invention, the linker peptide comprises an amino acidsequence having chemical and spatial properties similar to a peptideconsisting of SEQ ID NO:39. In certain aspects of the invention, thelinker peptide comprises SEQ ID NO:39, or conservative variants thereof.In certain aspects of the invention, the linker peptide comprises SEQ IDNO:38. In certain aspects of the invention, the linker peptide consistsof SEQ ID NO:39.

In certain aspects of the invention, the second amino acid sequencecomprises a sequence at least 80% identical, at least 85% identical, atleast 90% identical, at least 95% identical, at least 97% identical orat least 99% identical to a sequence selected from the group consistingof SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43. In certainaspects of the invention, the second amino acid sequence comprises asequence selected from the group consisting of SEQ ID NO:40, SEQ IDNO:41, SEQ ID NO:42, and SEQ ID NO:43.

One embodiment of the present invention is a protein construct (alsoreferred to as a fusion protein) comprising a first amino acid sequencefrom the stem region of a Group 2 influenza virus HA protein, a secondamino acid sequence from the stem region of a Group 2 influenza virus HAprotein, and a third amino acid sequence from the stem region of a Group2 influenza virus HA protein;

wherein the first amino acid sequence comprises at least 20 contiguousamino acid residues from the amino acid sequence upstream of theamino-terminal end of the head region sequence of an influenza A virusHA protein, or an amino acid sequence at least 85% identical, at least90% identical, at least 95% identical, at least 97% identical, or atleast 99% identical, to at least 40 contiguous amino acids from theamino acid sequence upstream of the amino-terminal end of the headregion sequence of an influenza A virus HA protein;

wherein the second amino acid sequence comprises at least 20 contiguousamino acid residues from the amino acid sequence that connects thecarboxyl-terminal end of the head region sequence to the inter-helixregion of an influenza A virus HA protein, or an amino acid sequence atleast 85% identical, at least 90% identical, at least 95% identical, atleast 97% identical, or at least 99% identical, to at least 40contiguous amino acid residues from the amino acid sequence thatconnects the carboxyl-terminal end of the head region sequence to theinter-helix region of an influenza A virus HA protein;

wherein the third amino acid sequence comprises at least 20 contiguousamino acid residues from the amino acid sequence that connects thecarboxyl-terminal end of the inter-helix region to the transmembranedomain (TM) of an influenza A virus HA protein, or an amino acidsequence at least 85% identical, at least 90% identical, at least 95%identical, at least 97% identical, or at least 99% identical, to atleast 40 contiguous amino acid residues from the amino acid sequencethat connects the carboxyl-terminal end of the inter-helix region to thetransmembrane domain of an influenza A virus HA protein;

wherein the first and second amino acid sequences are joined by a linkersequence; wherein the second and third amino acid sequences are joinedby a linker peptide; and,

wherein the first or third amino acid sequence is joined to at least aportion of a monomeric subunit domain such that the protein construct iscapable of forming a nanoparticle.

In certain aspects of the invention, the first amino acid sequence isfrom a Group 2 influenza HA protein. In one embodiment, the first aminoacid sequence is from a Group 2 influenza HA protein from a virusselected from the group consisting of an influenza H3 virus HA protein,an influenza H4 virus HA protein, an H7 influenza virus HA protein, anH10 influenza virus HA protein HA protein, an H14 influenza virus HAprotein, and an H15 influenza virus HA protein. In certain aspects ofthe invention, the first amino acid sequence is from a Group 2 influenzaHA protein from a Group 2 virus listed in Table 2. In certain aspects ofthe invention, the first amino acid sequence is from the stem region ofa Group 2 influenza HA protein having an amino acid sequences at least85%, at least 90%, at least 95% or at least 97% identical to a sequenceselected from the group consisting of SEQ ID NO:4-SEQ ID NO:26 and SEQID NO:47-159. In certain aspects of the invention, the first amino acidsequence is from the stem region of a Group 2 influenza HA proteincomprising a sequence selected from the group consisting of SEQ IDNO:4-SEQ ID NO:26 and SEQ ID NO:47-159.

In certain aspects of the invention, the first amino acid sequencecomprises at least 20 contiguous amino acid residues from the region ofa Group 2 influenza virus HA protein corresponding to amino acidresidues 1-59 of the HA protein of influenza A Denmark/35/2005 (H3N2)).In certain aspects of the invention, the first amino acid sequencecomprises at least 20 contiguous amino acid residues from a sequence atleast 85%, at least 90%, at least 95% or at least 97% identical to asequence selected from the group consisting of SEQ ID NO:27, SEQ IDNO:28 and SEQ ID NO:29. In certain aspects of the invention, the firstamino acid sequence comprises at least 20 contiguous amino acid residuesfrom a sequence selected from the group consisting of SEQ ID NO:27, SEQID NO:28 and SEQ ID NO:29.

In certain aspects of the invention, the first amino acid sequencecomprises at least 40 contiguous amino acid residues from the amino acidregion of an HA protein corresponding to amino acid residues 1-59 ofinfluenza A Denmark/35/2005 (H3N2)). In certain aspects of theinvention, the first amino acid sequence comprises at least 40contiguous amino acid residues from a sequence at least 85%, at least90%, at least 95% or at least 97% identical to SEQ ID NO:27 and SEQ IDNO:28. In certain aspects of the invention, the first amino acidsequence comprises at least 40 contiguous amino acid residues from SEQID NO:27 and SEQ ID NO:28.

In certain aspects of the invention, the first amino acid sequencecomprises a sequence corresponding to amino acid residues 1-59 ofinfluenza A Denmark/35/2005 (H3N2)) HA protein (SEQ ID NO:4). In certainaspects of the invention, the first amino acid sequence comprises asequence at least 85%, at least 90%, at least 95% or at least 97%identical to SEQ ID NO:27. In certain aspects of the invention, thefirst amino acid sequence comprises SEQ ID NO:27. In certain aspects ofthe invention, the first amino acid sequence consists of SEQ ID NO:27.

In certain aspects of the invention, the second amino acid sequence isfrom a Group 2 influenza HA protein. In certain aspects of theinvention, the second amino acid sequence is from a Group 2 influenza HAprotein from a virus selected from the group consisting of an influenzaH3 virus HA protein, an influenza H4 virus HA protein, an H7 influenzavirus HA protein, an H10 influenza virus HA protein HA protein, an H14influenza virus HA protein, and an H15 influenza virus HA protein. Incertain aspects of the invention, the second amino acid sequence is froma Group 2 influenza HA protein from a Group 2 virus listed in Table 2.In certain aspects of the invention, the second amino acid sequence isfrom the stem region of a Group 2 influenza HA protein having an aminoacid sequences at least 85%, at least 90%, at least 95% or at least 97%identical to a sequence selected from the group consisting of SEQ IDNO:4-SEQ ID NO:26 and SEQ ID NO:47-159. In certain aspects of theinvention, the second amino acid sequence is from the stem region of aGroup 2 influenza HA protein comprising a sequence selected from thegroup consisting of SEQ ID NO:4-SEQ ID NO:26 and SEQ ID NO:47-159.

In certain aspects of the invention, the second amino acid sequencecomprises at least 20 contiguous amino acids from a region of a Group 2influenza HA protein corresponding to amino acid residues 330-401 ofinfluenza A (Denmark/35/2005(H3N2)) (SEQ ID NO:4). In certain aspects ofthe invention, the second amino acid sequence comprises at least 20contiguous amino acid residues from a sequence at least 85%, at least90%, at least 95% or at least 97% identical to a sequence selected fromthe group consisting of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQID NO:33. In certain aspects of the invention, the second amino acidsequence comprises at least 20 contiguous amino acid residues from asequence selected from the group consisting of SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:32 and SEQ ID NO:33. In certain aspects of theinvention, the second amino acid sequence comprises at least 40contiguous amino acid residues from a sequence at least 85%, at least90%, at least 95% or at least 97% identical to a sequence selected fromthe group consisting of SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32. Incertain aspects of the invention, the second amino acid sequencecomprises at least 40 contiguous amino acid residues from a sequenceselected from the group consisting of SEQ ID NO:30, SEQ ID NO:31, andSEQ ID NO:32.

In certain aspects of the invention, the second amino acid sequencecomprises an amino acid sequence at least 85%, at least 90%, at least95% or at least 97% identical to SEQ ID NO:31. In certain aspects of theinvention, the second amino acid sequence comprises SEQ ID NO:31.

In certain aspects of the invention, the second amino acid sequencecomprises at least 60, or at least 72, contiguous amino acids from theamino acid sequence of a Group 2 influenza HA protein, that isimmediately downstream of the carboxyl-terminal end of the head regionsequence of the HA protein. In certain aspects of the invention, thesecond amino acid sequence comprises at least 60, or at least 72contiguous amino acids from the amino acid region of a Group 2 influenzavirus HA protein, that corresponds to amino acid residues 330-401 of aninfluenza A (Denmark/35/2005 (H3N2)) HA protein (SEQ ID NO:4).

The first and second amino acid sequences are connected by a linkersequence. In certain aspects of the invention, the linker sequence isless than 10 amino acids, less than 7 amino acids or less than 5 aminoacids in length. In certain aspects of the invention, the linkersequence comprises glycine and serine. In certain aspects of theinvention, the linker sequence joins the carboxyl-terminal end of thefirst amino acid sequence to the amino-terminal end of the second aminoacid sequence. In certain aspects of the invention, the linker sequencejoins the carboxyl-terminal end of the second amino acid sequence to theamino-terminal end of the first amino acid sequence. In certain aspectsof the invention, the linker sequence is similar in chemical and specialproperties to a peptide consisting of SEQ ID NO:34 or SEQ ID NO:35. Incertain aspects of the invention, the linker comprises SEQ ID NO:34 orSEQ ID NO:35, or conservative variants thereof. In one embodiment, thelinker comprises SEQ ID NO:34 or SEQ ID NO:35. In certain aspects of theinvention, the linker consists of SEQ ID NO:34 or SEQ ID NO:35.

In certain aspects of the invention, the third amino acid sequence isfrom a Group 2 influenza HA protein. In certain aspects of theinvention, the third amino acid sequence is from a Group 2 influenza HAprotein from a virus selected from the group consisting of an influenzaH3 virus HA protein, an influenza H4 virus HA protein, an H7 influenzavirus HA protein, an H10 influenza virus HA protein HA protein, an H14influenza virus HA protein, and an H15 influenza virus HA protein. Incertain aspects of the invention, the third amino acid sequence is froma Group 2 influenza HA protein from a Group 2 virus listed in Table 2.In certain aspects of the invention, the third amino acid sequence isfrom the stem region of a Group 2 influenza HA protein having an aminoacid sequences at least 85%, at least 90%, at least 95% or at least 97%identical to a sequence selected from the group consisting of SEQ IDNO:4-SEQ ID NO:26 and SEQ ID NO:47-159. In certain aspects of theinvention, the third amino acid sequence is from the stem region of aGroup 2 influenza HA protein comprising a sequence selected from thegroup consisting of SEQ ID NO:4-SEQ ID NO:26, and SEQ ID NO:47-159.

In certain aspects of the invention, the third amino acid sequencecomprises at least 20 contiguous amino acids from a region of a Group 2influenza HA protein corresponding to amino acid residues 438-519 ofinfluenza A (Denmark/35/2005(H3N2)) HA protein (SEQ ID NO:4). In certainaspects of the invention, the second amino acid sequence comprises atleast 20 contiguous amino acid residues from a sequence at least 85%, atleast 90%, at least 95% or at least 97% identical to a sequence selectedfrom the group consisting of SEQ ID NO:44, SEQ ID NO:45 and SEQ IDNO:46. In certain aspects of the invention, the third amino acidsequence comprises at least 20 contiguous amino acid residues from asequence selected from the group consisting of SEQ ID NO:44, SEQ IDNO:45 and SEQ ID NO:46. In certain aspects of the invention, the thirdamino acid sequence comprises at least 40 contiguous amino acid residuesfrom a sequence at least 85%, at least 90%, at least 95% or at least 97%identical to a sequence selected from the group consisting of SEQ IDNO:44, SEQ ID NO:45 and SEQ ID NO:46. In certain aspects of theinvention, the third amino acid sequence comprises at least 40contiguous amino acid residues from a sequence selected from the groupconsisting of SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46.

In certain aspects of the invention, the third amino acid sequencecomprises an amino acid sequence at least 85%, at least 90% at least 95%or at least 97% identical to a sequence selected from the groupconsisting of SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46. In certainaspects of the invention, the third amino acid sequence comprises anamino acid sequence selected from the group consisting of SEQ ID NO:44,SEQ ID NO:45, and SEQ ID NO:46.

In certain aspects of the invention, the third amino acid sequencecomprises at least 60, or at least 75, contiguous amino acids from theamino acid sequence of a Group 2 influenza HA protein, that isimmediately downstream of the carboxyl-terminal end of the inter-helixregion sequence of a Group 2 influenza A (Denmark/35/2005 (H3N2)) HAprotein. In certain aspects of the invention, the second amino acidsequence comprises at least 60, or at least 75 contiguous amino acidsfrom the amino acid region of a Group 2 influenza virus HA protein, thatcorresponds to amino acid residues 438-519 of an influenza A(Denmark/35/2005 (H3N2)) HA protein (SEQ ID NO:4).

The linker peptide can comprise any sequence of amino acids, as long asthe protein construct is able to form the desired conformation. Incertain aspects of the invention, the linker peptide is less than 10amino acids, less than 7 amino acids or less than 5 amino acids inlength. In certain aspects of the invention, the linker peptide is fouramino acids in length. In certain aspects of the invention, the linkersequence comprises an amino acid selected from the group consisting ofglycine, serine, proline and aspartic acid. In certain aspects of theinvention, the linker peptide comprises SEQ ID NO:39. In certain aspectsof the invention, the linker peptide consists of SEQ ID NO:39.

As has been discussed, mutations to various locations in proteinconstructs of the invention can stabilize the three-dimensionalstructure of the protein constructs and/or nanoparticles comprising theconstruct. Thus, in certain aspects of the invention, the first aminoacid sequence comprises at least one mutation at an amino acid locationcorresponding to a location in SEQ ID NO:4 selected from the groupconsisting G39, T46, and T58. In certain aspects of the invention, thefirst amino acid sequence comprises at least one mutation selected fromthe group consisting of G39C, T46C, and N54H, T58L (numbering based onthe sequence of the influenza A (Denmark/35/2005) (H3N2)) HA protein).

In certain aspects of the invention, the second amino acid sequencecomprises at least one mutation at an amino acid location correspondingto a location in SEQ ID NO:4 selected from the group consisting of L331,N338, Q392, K396, L397 and L400. In certain aspects of the invention,the first amino acid sequence comprises at least one mutation selectedfrom the group consisting of L331K, N338C, Q392C, and L400V (numberingbased on the sequence of the influenza A (Denmark/35/2005) (H3N2)) HAprotein).

In certain aspects of the invention, the third amino acid sequencecomprises at least one mutation at an amino acid location correspondingto a location in SEQ ID NO:4 selected from the group consisting of S438,N440, E448, T452, and N461. In certain aspects of the invention, thefirst amino acid sequence comprises at least one mutation selected fromthe group consisting of S438C, N440L, E448L, T452V, and N461R (numberingbased on the sequence of the influenza A (Denmark/35/2005) (H3N2)) HAprotein).

As noted above, protein constructs of the invention can be joined to atleast a portion of a monomeric subunit protein such that the proteinconstruct is capable of forming a nanoparticle. In certain aspects ofthe invention, the at least a portion of the monomeric subunit proteinis joined to the third amino acid sequence. In a preferred embodiment,the at least a portion of the monomeric subunit protein is joined to thecarboxyl end of the third amino acid sequence. In certain aspects of theinvention, the portion comprises at least 50, at least 100 or at least150 amino acids from a monomeric subunit. In certain aspects of theinvention, the monomeric subunit is ferritin. In certain aspects of theinvention, the monomeric subunit is lumazine synthase. In certainaspects of the invention, the portion comprises at least 50, at least100 or at least 150 amino acids from SEQ ID NO:1, SEQ ID NO:2 or SEQ IDNO:3. In certain aspects of the invention, the monomeric subunitcomprises a sequence at least 85% identical, at least 90% identical orat least 95% identical to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. Incertain aspects of the invention, the monomeric subunit comprises asequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2and SEQ ID NO:3.

While the modifications made to the Group 2 influenza virus HA proteinsdisclosed herein have been described as separate embodiments, it shouldbe appreciated that all such modification may be contained in a singleprotein construct. For example, a protein construct could be made inwhich a first amino acid sequence is joined by a linker to a secondamino acid sequence, wherein the second amino acid sequence comprises anamino acid sequence from the region downstream of the carboxyl-terminalend of the head region of a group 2 influenza HA protein, but in whichthe inter-helix region corresponding to amino acids 402-437 of the Group2 influenza A (Denmark/35/2005) (H3N2)) HA protein has been replacedwith a linker peptide, and wherein one or more mutations have beenintroduced into the second amino acid sequence at a locationcorresponding to a location selected from the group consisting of L331,N338, K396, L397, L400, S438, N440, E448, T452, and N461, of the Group 2influenza A (Denmark/35/2005) (H3N2)) HA protein, in order to increasethe strength of the interaction between these amino acid residues in thefolded protein.

While the protein constructs described heretofore can be used to producenanoparticles capable of generating an immune response against one ormore influenza viruses, in some embodiments, it may be useful toengineer further mutations into the amino acid sequences of proteins ofthe present invention. For example, it may be useful to alter sites suchas enzyme recognition sites or glycosylation sites in the monomericsubunit protein, the trimerization domain, or linker sequences, in orderto give the protein beneficial properties (e.g., solubility, half-life,mask portions of the protein from immune surveillance). In this regard,it is known that the monomeric subunit of ferritin is not glycosylatednaturally. However, it can be glycosylated if it is expressed as asecreted protein in mammalian or yeast cells. Thus, in certain aspectsof the invention, potential N-linked glycosylation sites in the aminoacid sequences from the monomeric ferritin subunit are mutated so thatthe mutated ferritin subunit sequences are no longer glycosylated at themutated site. One such sequence of a mutated monomeric ferritin subunitis represented by SEQ ID NO:2. Further description of useful mutationsare disclosed in International Application No. PCT/US2015/032695.

In some instances, it may be desirable to block the production of animmune response against certain amino acid sequences in the proteinconstruct. This may be done by adding a glycosylation site near the siteto be blocked such that the glycans sterically hinder the ability of theimmune system to reach the blocked site. Thus, in certain aspects of theinvention, the sequence of the protein construct has been altered toinclude one or more glycosylation sites. Examples of such sites include,but are not limited to, Asn-X-Ser, Asn-X-Thr and Asn-X-Cys. In someinstances, the glycosylation site can be introduced into a linkersequence. Further examples of useful sites at which to introduceglycosylation sites include, but are not limited to, locations in Group2 influenza HA proteins corresponding to amino acids 45-47, or aminoacids 370-372 of the HA protein of influenza A New Caledonia/20/1999(H1). Methods of introducing glycosylation sites are known to thoseskilled in the art.

Proteins and protein constructs of the present invention are encoded bynucleic acid molecules of the present invention. In addition, they areexpressed by nucleic acid constructs of the present invention. As usedherein a nucleic acid construct is a recombinant expression vector,i.e., a vector linked to a nucleic acid molecule encoding a protein suchthat the nucleic acid molecule can affect expression of the protein whenthe nucleic acid construct is administered to, for example, a subject oran organ, tissue or cell. The vector also enables transport of thenucleic acid molecule to a cell within an environment, such as, but notlimited to, an organism, tissue, or cell culture. A nucleic acidconstruct of the present disclosure is produced by human intervention.The nucleic acid construct can be DNA, RNA or variants thereof. Thevector can be a DNA plasmid, a viral vector, or other vector. In certainaspects of the invention, a vector can be a cytomegalovirus (CMV),retrovirus, adenovirus, adeno-associated virus, herpes virus, vacciniavirus, poliovirus, sindbis virus, or any other DNA or RNA virus vector.In certain aspects of the invention, a vector can be a pseudotypedlentiviral or retroviral vector. In certain aspects of the invention, avector can be a DNA plasmid. In certain aspects of the invention, avector can be a DNA plasmid comprising viral components and plasmidcomponents to enable nucleic acid molecule delivery and expression.Methods for the construction of nucleic acid constructs of the presentdisclosure are well known. See, for example, Molecular Cloning: ALaboratory Manual, 3^(rd) edition, Sambrook et al. 2001 Cold SpringHarbor Laboratory Press, and Current Protocols in Molecular Biology,Ausubel et al. eds., John Wiley & Sons, 1994. In certain aspects of theinvention, the vector is a DNA plasmid, such as a CMV/R plasmid such asCMV/R or CMV/R 8 KB (also referred to herein as CMV/R 8 kb). Examples ofCMV/R and CMV/R 8 kb are provided herein. CMV/R is also described inU.S. Pat. No. 7,094,598 B2, issued Aug. 22, 2006.

As used herein, a nucleic acid molecule comprises a nucleic acidsequence that encodes a protein construct of the present invention. Anucleic acid molecule can be produced recombinantly, synthetically, orby a combination of recombinant and synthetic procedures. A nucleic acidmolecule of the disclosure can have a wild-type nucleic acid sequence ora codon-modified nucleic acid sequence to, for example, incorporatecodons better recognized by the human translation system. In certainaspects of the invention, a nucleic acid molecule can begenetically-engineered to introduce, or eliminate, codons encodingdifferent amino acids, such as to introduce codons that encode anN-linked glycosylation site. Methods to produce nucleic acid moleculesof the disclosure are known in the art, particularly once the nucleicacid sequence is known. It is to be appreciated that a nucleic acidconstruct can comprise one nucleic acid molecule or more than onenucleic acid molecule. It is also to be appreciated that a nucleic acidmolecule can encode one protein or more than one protein.

In certain aspects of the invention the nucleic acid molecule of theinvention encodes a protein construct of the invention. In certainaspects of the invention, a nucleic acid molecule encodes a protein atleast 80% identical, at least 85% identical, at least 90% identical, atleast 95% identical, at least 97% identical, at least 99% identical to aprotein construct listed in Table 2. In certain aspects of theinvention, a nucleic acid molecule encodes a protein comprising an aminoacid sequence at least 80% identical, at least 85% identical, at least90% identical, at least 95% identical, at least 97% identical, at least99% identical to a sequence selected from the group consisting of SEQ IDNO:47-159.

Also encompassed by the present invention are expression systems forproducing protein constructs of the present invention. In certainaspects of the invention, nucleic acid molecules of the presentinvention are operationally linked to a promoter. As used herein,operationally linked means that proteins encoded by the linked nucleicacid molecules can be expressed when the linked promoter is activated.Promoters useful for practicing the present invention are known to thoseskilled in the art. One embodiment of the present invention is arecombinant cell comprising a nucleic acid molecule of the presentinvention. One embodiment of the present invention is a recombinantvirus comprising a nucleic acid molecule of the present invention.

As indicated above, the recombinant production of the protein constructsof the present invention can be accomplished using any suitableconventional recombinant technology currently known in the field. Forexample, production of a nucleic acid molecule encoding a fusion proteincan be carried out in E. coli using a nucleic acid molecule encoding asuitable monomeric subunit protein, such as the Helicobacter pyloriferritin monomeric subunit, and fusing it to a nucleic acid moleculeencoding a suitable influenza protein disclosed herein. The constructmay then be transformed into protein expression cells, grown to suitablesize, and induced to produce the fusion protein.

As has been described, because protein constructs of the presentinvention comprise a monomeric subunit protein, they can self-assemble.According to the present invention, the supramolecule resulting fromsuch self-assembly is referred to as an HA expressing, monomericsubunit-based nanoparticle. For ease of discussion, the HA expressing,monomeric subunit-based nanoparticle will simply be referred to as a, orthe, nanoparticle (np). Nanoparticles of the present invention havesimilar structural characteristics as the nanoparticles of the monomericprotein from which they are made. For example, with regard to ferritin,a ferritin-based nanoparticle contains 24 subunits and has 432 symmetry.In the case of nanoparticles of the present invention, the subunits arethe protein constructs comprising a monomeric subunit (e.g., ferritin,lumazine synthase, etc.) joined to a Group 2 influenza virus HA protein.Such nanoparticles display at least a portion of the Group 2 influenzavirus HA protein on their surface as HA trimers. In such a construction,the HA trimer is accessible to the immune system and thus can elicit animmune response. Thus, one embodiment of the invention is a nanoparticlecomprising any protein construct disclosed or described herein. Oneembodiment of the present invention is a nanoparticle comprising aprotein construct of the present invention, wherein the proteinconstruct comprises amino acids from the stem region of a Group 2influenza virus HA protein joined to a monomeric subunit protein. Incertain aspects of the invention, the nanoparticle displays the Group 2influenza virus HA protein on its surface as a HA trimer. In certainaspects of the invention, the Group 2 influenza virus HA protein iscapable of eliciting protective antibodies to an influenza virus.

One embodiment of the invention is a nanoparticle comprising a proteinconstruct of the invention. In certain aspects of the invention, theprotein construct comprises a Group 2 influenza HA protein wherein thehead region of the Group 2 influenza HA protein has been replaced withan amino acid sequence comprising less than 5 contiguous amino acidresidues from the head region of an influenza HA protein. In certainaspects of the invention, the HA protein of the protein construct hasalso been altered by extending the length of helix A. In certain aspectsof the invention, the HA protein of the protein construct has also beenaltered by shortening the inter-helix region or replacing theinter-helix region with a linker sequence. In certain aspects of theinvention, the HA protein of the protein construct has also been alteredby mutating specific locations to stabilize the trimeric structure.Examples of suitable locations include, but are not limited to,locations corresponding to a location in SEQ ID NO:4 selected from thegroup consisting of L331, N338, K396, L397, L400, S438, N440, E448,T452, N461, G39, T46, N54 and T58, and wherein the protein construct iscapable of forming a nanoparticle. Methods of replacing the HA proteinhead region, extending helix A, shortening or replacing the inter-helixregion, and suitable site-specific mutations have been disclosed herein.In certain aspects of the invention, the nanoparticle comprises aprotein construct comprising a first amino acid sequence from the stemregion of a Group 2 influenza virus HA protein and a second amino acidsequence from the stem region of a Group 2 influenza virus HA protein,the first and second amino acid sequences being covalently linked by alinker sequence,

-   -   wherein the first amino acid sequence comprises at least 20        contiguous amino acid residues from the amino acid sequence        upstream of the amino-terminal end of the head region sequence;    -   wherein the second amino acid sequence comprises at least 20        contiguous amino acid residues from the amino acid sequence        downstream of the carboxyl-terminal end of the head region        sequence; and,    -   wherein the first or second amino acid sequence is joined to at        least a portion of a monomeric subunit domain such that the        protein construct is capable of forming a nanoparticle.

In certain aspects of the invention, the first amino acid sequence isfrom the stem region of a Group 2 influenza virus HA protein from avirus selected from the group consisting of an influenza H3 virus HAprotein, an influenza H4 virus HA protein, an H7 influenza virus HAprotein, an H10 influenza virus HA protein HA protein, an H14 influenzavirus HA protein, and an H15 influenza virus HA protein. In certainaspects of the invention, the first amino acid sequence is from the stemregion of an HA protein from a Group 2 virus listed in Table 2. Incertain aspects of the invention, the first amino acid sequence is fromthe stem region of a Group 2 influenza HA protein having an amino acidsequences at least 85%, at least 90%, at least 95% or at least 97%identical to a sequence selected from the group consisting of SEQ IDNO:4-SEQ ID NO:26 and SEQ ID NO:47-SEQ ID NO:159. In certain aspects ofthe invention, the first amino acid sequence is from the stem region ofa Group 2 influenza HA protein comprising a sequence selected from thegroup consisting of SEQ ID NO:4-SEQ ID NO:26 and SEQ ID NO:47-SEQ IDNO:159.

In certain aspects of the invention, the second amino acid sequence isfrom the stem region of a Group 2 influenza HA protein from a virusselected from the group consisting of an influenza H3 virus, aninfluenza H4 virus, an H7 influenza virus, an H10 influenza virus, anH14 influenza virus, and an H15 influenza virus. In certain aspects ofthe invention, the second amino acid sequence is from the stem region ofan HA protein from a Group 2 virus listed in Table 2. In certain aspectsof the invention, the second amino acid sequence is from the stem regionof a Group 2 influenza virus HA protein having an amino acid sequencesat least 85%, at least 90%, at least 95% or at least 97% identical to asequence selected from the group consisting of SEQ ID NO:4-SEQ ID NO:26and SEQ ID NO:47-SEQ ID NO:159. In certain aspects of the invention, thesecond amino acid sequence is from the stem region of a Group 2influenza virus HA protein comprising a sequence selected from the groupconsisting of SEQ ID NO:4-SEQ ID NO:26 and SEQ ID NO:47-SEQ ID NO:159.

As noted above, the first amino acid sequence comprises at least 20contiguous amino acid residues from the amino acid sequence upstream ofthe amino-terminal end of the head region sequence. According to thepresent invention, the term upstream refers to the entirety of the aminoacid sequence linked to the amino-terminal end of the first amino acidresidue of the head region. Preferred upstream sequences are those thatare immediately adjacent to the head region sequence. In certain aspectsof the invention, the amino-terminal end of the head region is locatedat the amino acid residue corresponding to Q60 of the HA protein ofinfluenza A (Denmark/35/2005 (H3N2)) HA protein (SEQ ID NO:4) In certainaspects of the invention, the first amino acid sequence comprises atleast 20 contiguous amino acid residues from the region of a Group 2influenza virus HA protein corresponding to amino acid residues 1-59 ofthe HA protein of influenza A Denmark/35/2005 (H3N2)) represented by SEQID NO:4. In certain aspects of the invention, the first amino acidsequence comprises at least 20 contiguous amino acid residues from asequence at least 85%, at least 90%, at least 95% or at least 97%identical to a sequence selected from the group consisting of SEQ IDNO:27, SEQ ID NO:28 and SEQ ID NO:29. In certain aspects of theinvention, the first amino acid sequence comprises at least 20contiguous amino acid residues from a sequence selected from the groupconsisting of SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29.

In certain aspects of the invention, the first amino acid sequencecomprises at least 40 contiguous amino acid residues from the amino acidregion of an HA protein corresponding to amino acid residues 1-59 ofinfluenza A Denmark/35/2005 (H3N2)) HA protein (SEQ ID NO:4). In certainaspects of the invention, the first amino acid sequence comprises atleast 40 contiguous amino acid residues from a sequence at least 85%, atleast 90%, at least 95% or at least 97% identical to SEQ ID NO:27 or SEQID NO:28. In certain aspects of the invention, the first amino acidsequence comprises at least 40 contiguous amino acid residues from SEQID NO:27 or SEQ ID NO:28.

In certain aspects of the invention, the first amino acid sequencecomprises a sequence at least 85%, at least 90%, at least 95% or atleast 97% identical to SEQ ID NO:27. In certain aspects of theinvention, the first amino acid sequence comprises SEQ ID NO:27.

As noted above, the second amino acid sequence comprises at least 20contiguous amino acid residues from the amino acid sequence downstreamof the carboxyl-terminal end of the head region sequence. According tothe present invention, the term downstream refers to the entirety of theamino acid sequence linked to the carboxyl-terminal amino acid residueof the head region. Preferred upstream sequences are those that areimmediately adjacent to the head region sequence. In certain aspects ofthe invention, the carboxyl-terminal end of the head region is locatedat the amino acid position corresponding to T329 of the HA protein ofinfluenza A (Denmark/35/2005(H3N2)) HA protein represented by SEQ IDNO:4. Thus, in certain aspects of the invention, the second amino acidsequence comprises at least 20 contiguous amino acids from a region of aGroup 2 influenza HA protein corresponding to amino acid residues330-519 of influenza A (Denmark/35/2005) (H3N2) HA protein. In certainaspects of the invention, the second amino acid sequence comprises atleast 20 contiguous amino acids from a region of a Group 2 influenza HAprotein comprising amino acid residues 330-519 of influenza A(Denmark/35/2005(H3N2)) (SEQ ID NO:4). In certain aspects of theinvention, the second amino acid sequence comprises at least 20contiguous amino acid residues from a sequence at least 85%, at least90%, at least 95% or at least 97% identical to a sequence selected fromthe group consisting of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQID NO:33. In certain aspects of the invention, the second amino acidsequence comprises at least 20 contiguous amino acid residues from asequence selected from the group consisting of SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:32, and SEQ ID NO:33.

In certain aspects of the invention, the second amino acid sequencecomprises at least 40 contiguous amino acids from a region of a Group 2influenza HA protein corresponding to amino acid residues 330-519 ofinfluenza A (Denmark/35/2005) (H3N2) HA protein. In certain aspects ofthe invention, the second amino acid sequence comprises at least 40contiguous amino acids from a region of a Group 2 influenza HA proteincomprising amino acid residues 330-519 of influenza A(Denmark/35/2005(H3N2)) (SEQ ID NO:4). In certain aspects of theinvention, the second amino acid sequence comprises at least 40contiguous amino acid residues from a sequence at least 85%, at least90%, at least 95% or at least 97% identical to a sequence selected fromthe group consisting of SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32. Incertain aspects of the invention, the second amino acid sequencecomprises at least 20 contiguous amino acid residues from a sequenceselected from the group consisting of SEQ ID NO:30, SEQ ID NO:31, andSEQ ID NO:32.

In certain aspects of the invention, the second amino acid sequencecomprises an amino acid sequence at least 85%, at least 90%, at least95% or at least 97% identical to SEQ ID NO:37. In certain aspects of theinvention, the second amino acid sequence comprises SEQ ID NO:37.

In certain aspects of the invention, the second amino acid sequencecomprises at least 60, at least 72, at least 75, at least 100, at least150, at least 175, or at least 190 contiguous amino acids from a regionof a Group 2 influenza HA protein corresponding to amino acid residues330-519 of influenza A (Denmark/35/2005) (H3N2) HA protein. In certainaspects of the invention, the second amino acid sequence comprises atleast 60, at least 72, at least 75, at least 100, at least 150, at least175, or at least 190 contiguous amino acids from a region of a Group 2influenza HA protein comprising amino acid residues 330-519 of influenzaA (Denmark/35/2005(H3N2)) (SEQ ID NO:4). In certain aspects of theinvention, the second amino acid sequence comprises at least 40, atleast 60, at least 72, at least 75, at least 100, at least 150, at least175, or at least 190 contiguous amino acid residues from a sequence atleast 85%, at least 90%, at least 95% or at least 97% identical to SEQID NO:30. In certain aspects of the invention, the second amino acidsequence comprises at least 40, at least 60, at least 72, at least 75,at least 100, at least 150, at least 175, or at least 190 contiguousamino acid residues from SEQ ID NO:30.

In certain aspects of the invention, the nanoparticle comprises aprotein construct comprising an amino acid sequence at least 80%, atleast about 85%, at least about 90%, at least about 95%, at least about97% or at least about 99% identical to a protein construct sequencerecited in Table 2, wherein the nanoparticle is capable of selectivelybinding anti-influenza antibodies. In certain aspects of the invention,the nanoparticle comprises a protein construct comprising an amino acidsequence at least 80%, at least about 85%, at least about 90%, at leastabout 95%, at least about 97% or at least about 99% identical to asequence selected from the group consisting of SEQ ID NO:47-159, whereinthe nanoparticle is capable of selectively binding anti-influenzaantibodies. In certain aspects of the invention, the nanoparticlecomprises a protein construct comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 47-159.

Nanoparticles of the present invention can be used to elicit an immuneresponse to influenza virus. One type of immune response is a B-cellresponse, which results in the production of antibodies against theantigen that elicited the immune response. Thus, in certain aspects ofthe invention the nanoparticle elicits antibodies that bind to the stemregion of an influenza A HA protein from a virus selected from the groupconsisting of influenza A viruses, influenza B viruses and influenza Cviruses. One embodiment of the present invention is a nanoparticle thatelicits antibodies that bind to the stem region of influenza HA proteinselected from the group consisting of an H1 influenza virus HA protein,an H2 influenza virus HA protein, an influenza H3 virus HA protein, aninfluenza H4 virus HA protein, an influenza H5 virus HA protein, aninfluenza H6 virus HA protein, an H7 influenza virus HA protein, an H8influenza virus HA protein, an H9 influenza virus HA protein, an H10influenza virus HA protein HA protein, an H11 influenza virus HAprotein, an H12 influenza virus HA protein, an H13 influenza virus HAprotein, an H14 influenza virus HA protein, an H15 influenza virus HAprotein, an H16 influenza virus HA protein, an H17 influenza virus HAprotein, and an H18 influenza virus HA protein. One embodiment of thepresent invention is a nanoparticle that elicits antibodies that bind tothe stem region of an influenza HA protein from a virus listed in Table2.

While all antibodies are capable of binding to the antigen whichelicited the immune response that resulted in antibody production,preferred antibodies are those that provide broad heterosubtypicprotection against influenza virus. Thus, one embodiment of the presentinvention is a nanoparticle that elicits protective antibodies that bindto the stem region of influenza HA protein from a virus selected fromthe group consisting of influenza A viruses, influenza B viruses andinfluenza C viruses. One embodiment of the present invention is ananoparticle that elicits protective antibodies that bind to the stemregion of influenza HA protein selected from the group consisting of anH1 influenza virus HA protein, an H2 influenza virus HA protein, aninfluenza H3 virus HA protein, an influenza H4 virus HA protein, aninfluenza H5 virus HA protein, an influenza H6 virus HA protein, an H7influenza virus HA protein, an H8 influenza virus HA protein, an H9influenza virus HA protein, an H10 influenza virus HA protein HAprotein, an H11 influenza virus HA protein, an H12 influenza virus HAprotein, an H13 influenza virus HA protein, an H14 influenza virus HAprotein, an H15 influenza virus HA protein, an H16 influenza virus HAprotein, an H17 influenza virus HA protein, and an H18 influenza virusHA protein. One embodiment of the present invention is a nanoparticlethat elicits antibodies that bind to the stem region of an influenza HAprotein from a virus listed in Table 2. One embodiment of the presentinvention is a nanoparticle that elicits antibodies that bind to aprotein comprising an amino acid sequence at least 80% identical to asequence selected from the group consisting of SEQ ID NOs: 4-26. Oneembodiment of the present invention is a nanoparticle that elicitsantibodies that bind to a protein comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 4-26.

Protective antibodies elicited by proteins of the present invention canprotect against viral infections by affecting any step in the life cycleof the virus. For example, protective antibodies may prevent aninfluenza virus from attaching to a cell, entering a cell, releasingviral ribonucleoproteins into the cytoplasm, forming new viral particlesin the infected cell and budding new viral particles from the infectedhost cell membrane. In certain aspects of the invention, protectiveantibodies elicited by proteins of the present invention preventinfluenza virus from entering the host cell. In certain aspects of theinvention, protective antibodies elicited by proteins of the presentinvention prevent fusion of viral membranes with endosomal membranes. Incertain aspects of the invention, protective antibodies elicited byproteins of the present invention prevent release of ribonucleoproteinsinto the cytoplasm of the host cell. In certain aspects of theinvention, protective antibodies elicited by proteins of the presentinvention prevent assembly of new virus in the infected host cell. Incertain aspects of the invention, protective antibodies elicited byproteins of the present invention prevent release of newly formed virusfrom the infected host cell.

Because the amino acid sequence of the stem region of influenza virus ishighly conserved, protective antibodies elicited by nanoparticles of thepresent invention may be broadly protective. That is, protectiveantibodies elicited by nanoparticles of the present invention mayprotect against influenza viruses of more than one type, subtype and/orstrain. Thus, one embodiment of the present invention is a nanoparticlethat elicits broadly protective antibodies that bind the stem region ofinfluenza HA protein. One embodiment is a nanoparticle that elicitsantibodies that bind the stem region of an HA protein from more than onetype of influenza virus selected from the group consisting of influenzatype A viruses, influenza type B viruses and influenza type C viruses.One embodiment is a nanoparticle that elicits antibodies that bind thestem region of an HA protein from more than one sub-type of influenzavirus selected from the group consisting of an H1 influenza virus, an H2influenza virus, an influenza H3 virus, an influenza H4 virus, aninfluenza H5 virus, an influenza H6 virus, an H7 influenza virus, an H8influenza virus, an H9 influenza virus, an H10 influenza virus, an H11influenza virus, an H12 influenza virus, an H13 influenza virus, an H14influenza virus, an H15 influenza virus, an H16 influenza virus, an H17influenza virus, and an H18 influenza virus. One embodiment is ananoparticle that elicits antibodies that bind the stem region of an HAprotein from more than strain of influenza virus. One embodiment of thepresent invention is a nanoparticle that elicits antibodies that bindmore than one protein comprising an amino acid sequence at least 80%identical to a sequence selected from the group consisting of SEQ IDNO:4-SEQ ID NO:26. One embodiment of the present invention is ananoparticle that elicits antibodies that bind to more than one proteincomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 4-26.

As noted above, the HA sequence is linked to a portion of a monomericsubunit protein. As used herein, a monomeric subunit protein refers to aprotein monomer that is capable of binding to other monomeric subunitproteins such that the monomeric subunit proteins self-assemble into ananoparticle. Any monomeric subunit protein can be used to produce theprotein construct of the present invention, so long as the proteinconstruct is capable of forming a multimeric structure displaying HAprotein on its surface. In certain aspects of the invention themonomeric subunit is ferritin.

Ferritin is a globular protein found in all animals, bacteria, andplants, that acts primarily to control the rate and location ofpolynuclear Fe(III)₂O₃ formation through the transportation of hydratediron ions and protons to and from a mineralized core. The globular formof ferritin is made up of monomeric subunit proteins (also referred toas monomeric ferritin subunits), which are polypeptides having amolecule weight of approximately 17-20 kDa. An example of the sequenceof one such monomeric ferritin subunit is represented by SEQ ID NO 1.Each monomeric ferritin subunit has the topology of a helix bundle whichincludes a four antiparallel helix motif, with a fifth shorter helix(the c-terminal helix) lying roughly perpendicular to the long axis ofthe 4 helix bundle. According to convention, the helices are labeled A,B, C, and D & E from the N-terminus respectively. The N-terminalsequence lies adjacent to the nanoparticle three-fold axis and extendsto the surface, while the E helices pack together at the four-fold axiswith the C-terminus extending into the particle core. The consequence ofthis packing creates two pores on the nanoparticle surface. It isexpected that one or both of these pores represent the point by whichthe hydrated iron diffuses into and out of the nanoparticle. Followingproduction, these monomeric ferritin subunit proteins self-assemble intothe globular ferritin protein. Thus, the globular form of ferritincomprises 24 monomeric, ferritin subunit proteins, and has a capsid-likestructure having 432 symmetry.

According to the present invention, a monomeric ferritin subunit of thepresent invention is a full length, single polypeptide of a ferritinprotein, or any portion thereof, which is capable of directingself-assembly of monomeric ferritin subunits into the globular form ofthe protein. Examples of such proteins include, but are not limited toSEQ ID NO:1 and SEQ ID NO:2. Amino acid sequences from monomericferritin subunits of any known ferritin protein can be used to produceprotein constructs of the present invention, so long as the monomericferritin subunit is capable of self-assembling into a nanoparticledisplaying HA on its surface. In certain aspects of the invention, themonomeric subunit is from a ferritin protein selected from the groupconsisting of a bacterial ferritin protein, a plant ferritin protein, analgal ferritin protein, an insect ferritin protein, a fungal ferritinprotein and a mammalian ferritin protein. In certain aspects of theinvention, the ferritin protein is from Helicobacter pylori.

Protein constructs of the present invention need not comprise thefull-length sequence of a monomeric subunit polypeptide of a ferritinprotein. Portions, or regions, of the monomeric ferritin subunit proteincan be utilized so long as the portion comprises an amino acid sequencethat directs self-assembly of monomeric ferritin subunits into theglobular form of the protein. One example of such a region is locatedbetween amino acids 5 and 167 of the Helicobacter pylori ferritinprotein. More specific regions are described in Zhang, Y. Self-Assemblyin the Ferritin Nano-Cage Protein Super Family. 2011, Int. J. Mol. Sci.,12, 5406-5421, which is incorporated herein by reference in itsentirety.

In certain aspects of the invention the Group 2 influenza virus HAprotein is joined to at least 50, at least 100 or least 150 amino acidsfrom ferritin, wherein the protein construct is capable of forming ananoparticle. In certain aspects of the invention the Group 2 influenzavirus HA protein is joined to at least 50, at least 100 or least 150amino acids from SEQ ID NO:1 or SEQ ID NO:2, wherein the proteinconstruct is capable of forming a nanoparticle. In certain aspects ofthe invention the Group 2 influenza virus HA protein is joined to aprotein comprising an amino acid sequence at least 85%, at least 90% orat least 95% identical to the sequence of ferritin, wherein the proteinconstruct is capable of forming a nanoparticle. In certain aspects ofthe invention the Group 2 influenza virus HA protein is joined to aprotein comprising an amino acid sequence at least 85%, at least 90%, atleast 95% identical to SEQ ID NO:1 or SEQ ID NO:2, wherein the proteinconstruct is capable of forming a nanoparticle.

In certain aspects of the invention the monomeric subunit is lumazinesynthase. In certain aspects of the invention the Group 2 influenzavirus HA protein is joined to at least 50, at least 100 or least 150amino acids from lumazine synthase, wherein the protein construct iscapable of forming a nanoparticle. Thus, in certain aspects of theinvention the Group 2 influenza virus HA protein is joined to a proteinat least 85%, at least 90%, at least 95% identical to lumazine synthase,wherein the protein construct is capable of forming a nanoparticle.

As used herein, a nanoparticle of the present invention refers to athree-dimensional particle formed by self-assembly of protein constructs(fusion proteins) of the present invention. Nanoparticles of the presentinvention are generally spheroid in shape, although other shapes are notexcluded, and are generally from about 20 nm to about 100 nm indiameter. Nanoparticles of the present invention may, but need not,comprise other molecules, such as proteins, lipids, carbohydrates, etc.,than the protein constructs from which they are formed.

Because nanoparticles of the present invention can elicit an immuneresponse to an influenza virus, they are useful as vaccines to protectindividuals against infection by influenza virus. Thus, one embodimentof the present invention is a vaccine comprising a nanoparticle of thepresent invention. Vaccines of the present invention can also containother components such as adjuvants, buffers and the like. Although anyadjuvant can be used, preferred embodiments can contain: chemicaladjuvants such as aluminum phosphate, benzyalkonium chloride, ubenimex,and QS21; genetic adjuvants such as the IL-2 gene or fragments thereof,the granulocyte macrophage colony-stimulating factor (GM-CSF) gene orfragments thereof, the IL-18 gene or fragments thereof, the chemokine(C-C motif) ligand 21 (CCL21) gene or fragments thereof, the IL-6 geneor fragments thereof, CpG, LPS, TLR agonists, and other immunestimulatory genes; protein adjuvants such IL-2 or fragments thereof, thegranulocyte macrophage colony-stimulating factor (GM-CSF) or fragmentsthereof, IL-18 or fragments thereof, the chemokine (C-C motif) ligand 21(CCL21) or fragments thereof, IL-6 or fragments thereof, CpG, LPS, TLRagonists and other immune stimulatory cytokines or fragments thereof;lipid adjuvants such as cationic liposomes, N3 (cationic lipid),monophosphoryl lipid A (MPL1); other adjuvants including cholera toxin,enterotoxin, Fms-like tyrosine kinase-3 ligand (Flt-3L), bupivacaine,marcaine, and alevamisole.

One embodiment of the present invention is a nanoparticle vaccine thatincludes more than one influenza HA protein. Such a vaccine can includea combination of different influenza HA proteins, either on a singlenanoparticle or as a mixture of nanoparticles, at least two of whichhave unique influenza HA proteins. A multivalent vaccine can comprise asmany influenza HA proteins as necessary in order to result in productionof the immune response necessary to protect against a desired breadth ofvirus strains. In certain aspects of the invention, the vaccinecomprises an HA protein from at least two different influenza strains(bi-valent). In certain aspects of the invention, the vaccine comprisesa HA protein from at least three different influenza strains(tri-valent). In certain aspects of the invention, the vaccine comprisesan HA protein from at least four different influenza strains(tetra-valent). In certain aspects of the invention, the vaccinecomprises an HA protein from at least five different influenza strains(penta-valent). In certain aspects of the invention, the vaccinecomprises an HA protein from at least six different influenza strains(hexa-valent). In various embodiments, a vaccine comprises an HA proteinfrom each of 7, 8, 9, or 10 different strains of influenza virus. Anexample of such a combination is a nanoparticle vaccine that comprisesinfluenza A group 1 HA protein, an influenza A group 2 HA protein, andan influenza B HA protein. In certain aspects of the invention, theinfluenza HA proteins are H1 HA, H3 HA, and B HA. Another example of amultivalent vaccine is a nanoparticle vaccine that comprises HA proteinsfrom four different influenza viruses. In certain aspects of theinvention, the multivalent vaccine comprises one or more HA proteins atleast 80% identical, at least 85% identical, at least 90% identical, atleast 95% identical, at least 97% identical or at least 99% identical toone or more HA proteins listed in Table 2. In certain aspects of theinvention, the multivalent vaccine comprises one or more HA proteinslisted in Table 2.

One embodiment of the present invention is a method to vaccinate anindividual against influenza virus, the method comprising administeringa nanoparticle to an individual such that an immune response againstinfluenza virus is produced in the individual, wherein the nanoparticlecomprises a monomeric subunit protein joined to a Group 2 influenzavirus HA protein, and wherein the nanoparticle displays the influenza HAon its surface. In certain aspects of the invention, the nanoparticle isa monovalent nanoparticle. In certain aspects of the invention, thenanoparticle is multivalent nanoparticle. Another embodiment of thepresent invention is a method to vaccinate an individual againstinfection with influenza virus, the method comprising:

a) obtaining a nanoparticle comprising monomeric subunits, wherein themonomeric subunits are joined to an influenza hemagglutinin protein, andwherein the nanoparticle displays a Group 2 influenza virus HA proteinon its surface; and,

b) administering the nanoparticle to an individual such that an immuneresponse against an influenza virus is produced.

One embodiment of the present invention is a method to vaccinate anindividual against influenza virus, the method comprising administeringa vaccine of the embodiments to an individual such that an immuneresponse against influenza virus is produced in the individual, whereinthe vaccine comprises at least one nanoparticle comprising a monomericsubunit joined to an influenza HA protein, and wherein the nanoparticledisplays the influenza HA on its surface. In certain aspects of theinvention, the vaccine is a monovalent vaccine. In certain aspects ofthe invention, the vaccine is multivalent vaccine. One embodiment of thepresent invention is a method to vaccinate an individual againstinfection with influenza virus, the method comprising:

a) obtaining a vaccine comprising at least one nanoparticle comprising aprotein construct of the present invention, wherein the proteinconstruct comprises a monomeric subunit protein joined to a Group 2influenza virus HA protein, and wherein the nanoparticle displays theinfluenza HA on its surface; and,

b) administering the vaccine to an individual such that an immuneresponse against an influenza virus is produced.

Certain aspects of the invention, the nanoparticle is a monovalentnanoparticle. Certain aspects of the invention, the nanoparticle ismultivalent nanoparticle.

Certain aspects of the invention, the nanoparticle has octahedralsymmetry. Certain aspects of the invention, the influenza HA protein iscapable of eliciting antibodies to an influenza virus. Certain aspectsof the invention, the influenza HA protein is capable of elicitingbroadly antibodies to an influenza virus. In preferred embodiments theelicited antibodies are protective antibodies. In a preferredembodiment, the elicited antibodies are broadly heterosubtypicprotective.

Vaccines of the present invention can be used to vaccinate individualsusing a prime/boost protocol. Such a protocol is described in U.S.Patent Publication No. 20110177122, which is incorporated herein byreference in its entirety. In such a protocol, a first vaccinecomposition may be administered to the individual (prime) and then aftera period of time, a second vaccine composition may be administered tothe individual (boost). Administration of the boosting composition isgenerally weeks or months after administration of the primingcomposition, preferably about 2-3 weeks or 4 weeks, or 8 weeks, or 16weeks, or 20 weeks, or 24 weeks, or 28 weeks, or 32 weeks. Certainaspects of the invention, the boosting composition is formulated foradministration about 1 week, or 2 weeks, or 3 weeks, or 4 weeks, or 5weeks, or 6 weeks, or 7 weeks, or 8 weeks, or 9 weeks, or 16 weeks, or20 weeks, or 24 weeks, or 28 weeks, or 32 weeks after administration ofthe priming composition

The first and second vaccine compositions can be, but need not be, thesame composition. Thus, certain aspects of the invention of the presentinvention, the step of administering the vaccine comprises administeringa first vaccine composition, and then at a later time, administering asecond vaccine composition. Certain aspects of the invention, the firstvaccine composition comprises a nanoparticle of the present invention.Certain aspects of the invention, the first vaccine compositioncomprises a nanoparticle of the invention.

Certain aspects of the invention, the individual being vaccinated hasbeen exposed to influenza virus. As used herein, the terms exposed,exposure, and the like, indicate the subject has come in contact with aperson of animal that is known to be infected with an influenza virus.Vaccines of the present invention may be administered using techniqueswell known to those in the art. Techniques for formulation andadministration may be found, for example, in “Remington's PharmaceuticalSciences”, 18th ed., 1990, Mack Publishing Co., Easton, Pa. Vaccines maybe administered by means including, but not limited to, traditionalsyringes, needleless injection devices, or micro-projectile bombardmentgene guns. Suitable routes of administration include, but are notlimited to, parenteral delivery, such as intramuscular, intradermal,subcutaneous, intramedullary injections, as well as, intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections, just to name a few. For injection, the compoundsof one embodiment of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks' solution, Ringer's solution, or physiological saline buffer.

Certain aspects of the invention, vaccines, or nanoparticles, of thepresent invention can be used to protect an individual against infectionby heterologous influenza virus. That is, a vaccine made using HAprotein from one strain of influenza virus is capable of protecting anindividual against infection by different strains of influenza. Forexample, a vaccine made using HA protein from influenzaA/Denmark/35/2005)(H3N2), can be used to protect an individual againstinfection by an influenza virus recited in Table 2.

Certain aspects of the invention, vaccines, or nanoparticles, of thepresent invention can be used to protect an individual against infectionby an antigenically divergent influenza virus. Antigenically divergentrefers to the tendency of a strain of influenza virus to mutate overtime, thereby changing the amino acids that are displayed to the immunesystem. Such mutation over time is also referred to as antigenic drift.Thus, for example, a vaccine made using HA protein from the influenzaA/Denmark/35/2005)(H3N2) strain of influenza virus is capable ofprotecting an individual against infection by earlier, antigenicallydivergent Denmark strains of influenza, and by evolving (or diverging)influenza strains of the future.

Because nanoparticles of the present invention display Group 2 influenzavirus HA proteins that are antigenically similar to an intact HA, theycan be used in assays for detecting antibodies against influenza virus(anti-influenza antibodies).

Thus, one embodiment of the present invention is a method for detectinganti-influenza virus antibodies using nanoparticles of the presentinvention. A detection method of the present invention can generally beaccomplished by:

a. contacting at least a portion of a sample being tested for thepresence of anti-influenza antibodies with a nanoparticle of the presentinvention; and,

b. detecting the presence of a nanoparticle/antibody complex;

wherein the presence of a nanoparticle/antibody complex indicates thatthe sample contains anti-influenza antibodies.

Certain aspects of the invention of the present invention, a sample isobtained, or collected, from an individual to be tested for the presenceof anti-influenza virus antibodies. The individual may or may not besuspected of having anti-influenza antibodies or of having been exposedto influenza virus. A sample is any specimen obtained from theindividual that can be used to test for the presence of anti-influenzavirus antibodies. A preferred sample is a body fluid that can be used todetect the presence of anti-influenza virus antibodies. Examples of bodyfluids that may be used to practice the present method include, but arenot limited to, blood, plasma, serum, lacrimal fluid and saliva. Thoseskilled in the art can readily identify samples appropriate forpracticing the disclosed methods.

Blood, or blood-derived fluids such as plasma, serum, and the like, areparticularly suitable as the sample. Such samples can be collected andprepared from individuals using methods known in the art. The sample maybe refrigerated or frozen before assay.

Any nanoparticle of the present invention can be used to practice thedisclosed method as long as the nanoparticle binds to anti-influenzavirus antibodies. Useful nanoparticles, and methods of their production,have been described in detail herein. In a preferred embodiment, thenanoparticle comprises a protein construct, wherein the proteinconstruct comprises at least 25, at least 50, at least 75, at least 100,or at least 150 contiguous amino acids from a monomeric subunit proteinjoined to (fused to) at least one epitope from a Group 2 influenza virusHA protein such that the nanoparticle comprises trimers of the Group 2influenza virus HA protein epitope on its surface, and wherein theprotein construct is capable of self-assembling into nanoparticles.

As used herein, the term contacting refers to the introduction of asample being tested for the presence of anti-influenza antibodies to ananoparticle of the present invention, for example, by combining ormixing the sample and the nanoparticle of the present invention, suchthat the nanoparticle is able to come into physical contact withantibodies in the sample, if present. When anti-influenza virusantibodies are present in the sample, an antibody/nanoparticle complexis then formed. Such complex formation refers to the ability of ananti-influenza virus antibodies to selectively bind to the HA portion ofthe protein construct in the nanoparticle in order to form a stablecomplex that can be detected. Binding of anti-influenza virus antibodiesin the sample to the nanoparticle is accomplished under conditionssuitable to form a complex. Such conditions (e.g., appropriateconcentrations, buffers, temperatures, reaction times) as well asmethods to optimize such conditions are known to those skilled in theart. Binding can be measured using a variety of methods standard in theart including, but not limited to, agglutination assays, precipitationassays, enzyme immunoassays (e.g., ELISA), immunoprecipitation assays,immunoblot assays and other immunoassays as described, for example, inSambrook et al., Molecular Cloning: A Laboratory Manual, (Cold SpringHarbor Labs Press, 1989), and Harlow et al., Antibodies, a LaboratoryManual (Cold Spring Harbor Labs Press, 1988), both of which areincorporated by reference herein in their entirety. These referencesalso provide examples of complex formation conditions.

As used herein, the phrases selectively binds HA, selective binding toHA, and the like, refer to the ability of an antibody to preferentiallybind a HA protein as opposed to binding proteins unrelated to HA, ornon-protein components in the sample or assay. An antibody thatselectively binds HA is one that binds HA but does not significantlybind other molecules or components that may be present in the sample orassay. Significant binding, is considered, for example, binding of ananti-HA antibody to a non-HA molecule with an affinity or avidity greatenough to interfere with the ability of the assay to detect and/ordetermine the level of, anti-influenza antibodies in the sample.Examples of other molecules and compounds that may be present in thesample, or the assay, include, but are not limited to, non-HA proteins,such as albumin, lipids and carbohydrates.

Certain aspects of the invention, an anti-influenza virusantibody/nanoparticle complex, also referred to herein as anantibody/nanoparticle complex, can be formed in solution. Certainaspects of the invention an antibody/nanoparticle complex can be formedin which the nanoparticle is immobilized on (e.g., coated onto) asubstrate. Immobilization techniques are known to those skilled in theart. Suitable substrate materials include, but are not limited to,plastic, glass, gel, celluloid, fabric, paper, and particulatematerials. Examples of substrate materials include, but are not limitedto, latex, polystyrene, nylon, nitrocellulose, agarose, cotton, PVDF(poly-vinylidene-fluoride), and magnetic resin. Suitable shapes forsubstrate material include, but are not limited to, a well (e.g.,microtiter dish well), a microtiter plate, a dipstick, a strip, a bead,a lateral flow apparatus, a membrane, a filter, a tube, a dish, acelluloid-type matrix, a magnetic particle, and other particulates.Particularly preferred substrates include, for example, an ELISA plate,a dipstick, an immunodot strip, a radioimmunoassay plate, an agarosebead, a plastic bead, a latex bead, a cotton thread, a plastic chip, animmunoblot membrane, an immunoblot paper and a flow-through membrane.Certain aspects of the invention, a substrate, such as a particulate,can include a detectable marker. For descriptions of examples ofsubstrate materials, see, for example, Kemeny, D. M. (1991) A PracticalGuide to ELISA, Pergamon Press, Elmsford, N.Y. pp 33-44, and Price, C.and Newman, D. eds. Principles and Practice of Immunoassay, 2nd edition(1997) Stockton Press, NY, N.Y., both of which are incorporated hereinby reference in their entirety.

In accordance with the present invention, once formed, an anti-influenzavirus antibody/nanoparticle complex is detected. Detection can bequalitative, quantitative, or semi-quantitative. As used herein, thephrases detecting complex formation, detecting the complex, and thelike, refer to identifying the presence of anti-influenza virus antibodycomplexed with the nanoparticle. If complexes are formed, the amount ofcomplexes formed can, but need not be, quantified. Complex formation, orselective binding, between a putative anti-influenza virus antibody anda nanoparticle can be measured (i.e., detected, determined) using avariety of methods standard in the art (see, for example, Sambrook etal. supra.), examples of which are disclosed herein. A complex can bedetected in a variety of ways including, but not limited to use of oneor more of the following assays: a hemagglutination inhibition assay, aradial diffusion assay, an enzyme-linked immunoassay, a competitiveenzyme-linked immunoassay, a radioimmunoassay, a fluorescenceimmunoassay, a chemiluminescent assay, a lateral flow assay, aflow-through assay, a particulate-based assay (e.g., using particulatessuch as, but not limited to, magnetic particles or plastic polymers,such as latex or polystyrene beads), an immunoprecipitation assay, aBioCoreJ assay (e.g., using colloidal gold), an immunodot assay (e.g.,CMG Immunodot System, Fribourg, Switzerland), and an immunoblot assay(e.g., a western blot), an phosphorescence assay, a flow-through assay,a chromatography assay, a PAGe-based assay, a surface plasmon resonanceassay, a spectrophotometric assay, and an electronic sensory assay. Suchassays are well known to those skilled in the art.

Assays can be used to give qualitative or quantitative results dependingon how they are used. Some assays, such as agglutination, particulateseparation, and precipitation assays, can be observed visually (e.g.,either by eye or by a machines, such as a densitometer orspectrophotometer) without the need for a detectable marker.

In other assays, conjugation (i.e., attachment) of a detectable markerto the nanoparticle, or to a reagent that selectively binds to thenanoparticle, aids in detecting complex formation. A detectable markercan be conjugated to the nanoparticle, or nanoparticle-binding reagent,at a site that does not interfere with ability of the nanoparticle tobind to an anti-influenza virus antibody. Methods of conjugation areknown to those of skill in the art. Examples of detectable markersinclude, but are not limited to, a radioactive label, a fluorescentlabel, a chemiluminescent label, a chromophoric label, an enzyme label,a phosphorescent label, an electronic label; a metal sol label, acolored bead, a physical label, or a ligand. A ligand refers to amolecule that binds selectively to another molecule. Preferreddetectable markers include, but are not limited to, fluorescein, aradioisotope, a phosphatase (e.g., alkaline phosphatase), biotin,avidin, a peroxidase (e.g., horseradish peroxidase), beta-galactosidase,and biotin-related compounds or avidin-related compounds (e.g.,streptavidin or ImmunoPure7 NeutrAvidin).

Certain aspects of the invention, an antibody/nanoparticle complex canbe detected by contacting a sample with a specific compound, such as anantibody, that binds to an anti-influenza antibody, ferritin, or to theantibody/nanoparticle complex, conjugated to a detectable marker. Adetectable marker can be conjugated to the specific compound in such amanner as not to block the ability of the compound to bind to thecomplex being detected. Preferred detectable markers include, but arenot limited to, fluorescein, a radioisotope, a phosphatase (e.g.,alkaline phosphatase), biotin, avidin, a peroxidase (e.g., horseradishperoxidase), beta-galactosidase, and biotin-related compounds oravidin-related compounds (e.g., streptavidin or ImmunoPure7NeutrAvidin).

In another embodiment, a complex is detected by contacting the complexwith an indicator molecule. Suitable indicator molecules includemolecules that can bind to the anti-influenza virusantibody/nanoparticle complex, the anti-influenza virus antibody, or thenanoparticle. As such, an indicator molecule can comprise, for example,a reagent that binds the anti-influenza virus antibody, such as anantibody that recognizes immunoglobulins. Preferred indicator moleculesthat are antibodies include, for example, antibodies reactive with theantibodies from species of individual in which the anti-influenza virusantibodies are produced. An indicator molecule itself can be attached toa detectable marker of the present invention. For example, an antibodycan be conjugated to biotin, horseradish peroxidase, alkalinephosphatase or fluorescein.

The present invention can further comprise one or more layers and/ortypes of secondary molecules or other binding molecules capable ofdetecting the presence of an indicator molecule. For example, anuntagged (i.e., not conjugated to a detectable marker) secondaryantibody that selectively binds to an indicator molecule can be bound toa tagged (i.e., conjugated to a detectable marker) tertiary antibodythat selectively binds to the secondary antibody. Suitable secondaryantibodies, tertiary antibodies and other secondary or tertiarymolecules can be readily selected by those skilled in the art. Preferredtertiary molecules can also be selected by those skilled in the artbased upon the characteristics of the secondary molecule. The samestrategy can be applied for subsequent layers.

Preferably, the indicator molecule is conjugated to a detectable marker.A developing agent is added, if required, and the substrate is submittedto a detection device for analysis. In some protocols, washing steps areadded after one or both complex formation steps in order to removeexcess reagents. If such steps are used, they involve conditions knownto those skilled in the art such that excess reagents are removed butthe complex is retained.

Because assays of the present invention can detect anti-influenza virusantibodies in a sample, including a blood sample, such assays can beused to identify individuals having anti-influenza antibodies. Thus, oneembodiment of the present invention is a method to identify anindividual having anti-influenza virus antibodies, the methodcomprising:

-   -   a. contacting a sample from an individual being tested for        anti-influenza antibodies with a nanoparticle of the present        invention; and,    -   b. analyzing the contacted sample for the presence of a        nanoparticle/antibody complex    -   wherein the presence of a nanoparticle/antibody complex        indicates the individual has anti-influenza antibodies.

Any of the disclosed assay formats can be used to conduct the disclosedmethod. Examples of useful assay formats include, but are not limitedto, a radial diffusion assay, an enzyme-linked immunoassay, acompetitive enzyme-linked immunoassay, a radioimmunoassay, afluorescence immunoassay, a chemiluminescent assay, a lateral flowassay, a flow-through assay, a particulate-based assay (e.g., usingparticulates such as, but not limited to, magnetic particles or plasticpolymers, such as latex or polystyrene beads), an immunoprecipitationassay, a BioCoreJ assay (e.g., using colloidal gold), an immunodot assay(e.g., CMG Immunodot System, Fribourg, Switzerland), and an immunoblotassay (e.g., a western blot), an phosphorescence assay, a flow-throughassay, a chromatography assay, a PAGe-based assay, a surface plasmonresonance assay, bio-layer interferometry assay, a spectrophotometricassay, and an electronic sensory assay.

If no anti-influenza antibodies are detected in the sample, such aresult indicates the individual does not have anti-influenza virusantibodies. The individual being tested may or may not be suspected ofhaving antibodies to influenza virus. The disclosed methods may also beused to determine if an individual has been exposed to one or morespecific type, group, sub-group or strain of influenza virus. To makesuch a determination, a sample is obtained from an individual that hastested negative for antibodies (i.e., lacked antibodies) to one or morespecific type, group, sub-group or strain of influenza virus sometime intheir past (e.g., greater than about 1 year, greater than about 2 years,greater than about 3 years, greater than about 4 years, greater thanabout 5 years, etc.). The sample is then tested for the presence ofanti-influenza virus antibodies to one or more type, group, sub-group orstrain, of influenza virus using a nanoparticle-based assay of thepresent invention. If the assay indicates the presence of suchantibodies, the individual is then identified as having been exposed toone or more type, group sub-group or strain, of influenza virus sometimeafter the test identifying them as negative for anti-influenzaantibodies. Thus, one embodiment of the present invention is method toidentify an individual that has been exposed to influenza virus, themethod comprising:

-   -   a. contacting at least a portion of a sample from an individual        being tested for anti-influenza antibodies with a nanoparticle        of the present invention; and,    -   b. analyzing the contacted sample for the presence or level of        an antibody/nanoparticle complex, wherein the presence or level        of antibody/nanoparticle complex indicates the presence or level        of recent anti-influenza antibodies;    -   c. comparing the recent anti-influenza antibody level with a        past anti-influenza antibody level;    -   wherein an increase in the recent anti-influenza antibody level        over the past anti-influenza antibody level indicates the        individual has been exposed to influenza virus subsequent to        determination of the past anti-influenza antibody level.

Methods of the present invention are also useful for determining theresponse of an individual to a vaccine. Thus, one embodiment is a methodfor measuring the response of an individual to an influenza vaccine, themethod comprising:

-   -   a. administering to the individual a vaccine for influenza        virus;    -   b. contacting at least a portion of a sample from the individual        with a nanoparticle of the present invention;    -   c. analyzing the contacted sample for the presence or level of        an antibody/nanoparticle complex, wherein the presence or level        of antibody/nanoparticle complex indicates the presence or level        of recent anti-influenza antibodies    -   wherein an increase in the level of antibody in the sample over        the pre-vaccination level of antibody in the individual        indicates the vaccine induced an immune response in the        individual.

The influenza vaccine administered to the individual may, but need not,comprise a vaccine of the present invention, as long as the nanoparticlecomprises an HA protein that can bind an anti-influenza antibody inducedby the administered vaccine. Methods of administering influenza vaccinesare known to those of skill in the art.

Analysis of the sample obtained from the individual may be performedusing any of the disclosed assay formats. Certain aspects of theinvention, analysis of the sample is performed using an assay formatselected from the group consisting of, a radial diffusion assay, anenzyme-linked immunoassay, a competitive enzyme-linked immunoassay, aradioimmunoassay, a fluorescence immunoassay, a chemiluminescent assay,a lateral flow assay, a flow-through assay, a particulate-based assay(e.g., using particulates such as, but not limited to, magneticparticles or plastic polymers, such as latex or polystyrene beads), animmunoprecipitation assay, a BioCoreJ assay (e.g., using colloidalgold), an immunodot assay (e.g., CMG Immunodot System, Fribourg,Switzerland), and an immunoblot assay (e.g., a western blot), anphosphorescence assay, a flow-through assay, a chromatography assay, aPAGE-based assay, a surface plasmon resonance assay, bio-layerinterferometry assay, a spectrophotometric assay, and an electronicsensory assay.

Certain aspects of the invention, the method includes a step ofdetermining the level of anti-influenza antibody present in theindividual prior to administering the vaccine. However, it is alsopossible to determine the level of anti-influenza antibody present inthe individual from prior medical records, if such information isavailable.

While not necessary to perform the disclosed method, it may bepreferable to wait some period of time between the step of administeringthe vaccine and the step of determining the level of anti-influenzaantibody in the individual. Certain aspects of the invention,determination of the level of anti-influenza antibodies present in theindividual is performed at least 1 day, at least 2 days, at least 3days, at least 4 days, at least 5 days, at least 6 days, at least oneweek, at least two weeks, at least three weeks, at least four weeks, atleast two months, at least three months or at least six months,following administration of the vaccine.

The present invention also includes kits suitable for detectinganti-influenza antibodies. Suitable means of detection include thetechniques disclosed herein, utilizing nanoparticles of the presentinvention. Kits may also comprise a detectable marker, such as anantibody that selectively binds to the nanoparticle, or other indicatormolecules. The kit can also contain associated components, such as, butnot limited to, buffers, labels, containers, inserts, tubings, vials,syringes and the like.

Examples

This example characterizes the properties and activities of five H10variants of Group 2 HA nanoparticles, designed using the parameters andmethodology disclosed herein. All of the variants were based on thehuman A/Jiangxi/IPB13/2013(H10N8) strain. Nucleic acid moleculesencoding the H10 variants were introduced into Expi293 cells, and thecells cultured under conditions suitable for expression of the encodedvariant proteins. Expressed nanoparticles were purified from cellculture supernatant using lectin affinity chromatography followed bysize exclusion chromatography (SEC). Chromatograms for the purifiednanoparticles are shown in FIGS. 32A-32E.

The purified nanoparticles were analyzed by negative stain electronmicroscopy, which indicated that individual nanoparticles were formedwith the HA stems projecting outward in a periodic arrangement. Arepresentative electron micrograph for each variant is show in FIG. 33.

The antigenicity of the H10ssF variants was evaluated in an ELISA formatby measuring affinity to HA stem antibodies FI6, CT149 and CR8020. Theresults of this evaluation are shown in FIGS. 34A-34D.

The nanoparticles were then tested for their ability to elicit an immuneresponse against various influenza strains in mice. BALB/c mice (n=10)were immunized with 2 ug of one of the variant nanoparticles using SASadjuvant. The immunization was repeated 2 more times at periodicintervals. 2 weeks after the last immunization, sera was collected andtested (by ELISA) for its ability to recognize HA protein from H3N2 andH7N9. The results, which are illustrated in FIGS. 35A & 35B, demonstratethat the sera was cross-reactive for both H3N2 and H7N9 HA protein.

The immunized mice were then challenged with a lethal dose of H3N2(A/Philippines/1982) or H7N9 (A/Shanghai/2/2013-like), and weight lossand survival monitored. The results, which are shown in FIGS. 36A-36Dand FIGS. 37A-37G, showed that immunization with the variantsnanoparticles protected against both challenge strains withoutsignificant weight loss. These results demonstrate that H10ssFimmunogens can provide heterosubtypic protection against H3N2 and H7N9strains.

It has been shown that the human, broadly neutralizing stem monoclonalantibody (mAb) 16.a.26), which uses a VH1-18 v-gene, can potentlyneutralize both group 1 and group 2 influenza viruses. Thus, severalHA-SS-np variants, including H3N2, H7N9 and H10N8 subtypes, wereevaluated for their ability to activate B cells expressing agermline-reverted version of mAb 16.a.26. In the assay, activation ofB-cells is indicated by Ca++ flux. The results of this evaluation, whichare shown in FIG. 40, show that the variant nanoparticles H3ssF_256,H7ssF_26 and H10ssF_04 each resulted high levels of activation similarto that observed by the IgM positive control. As shown in FIG. 41, allthree of these designs share the same helix A C-terminal extension(ELMEQ), suggesting that this particular motif is useful for eliciting a16.a.26 bNAb response against influenza HA proteins.

What is claimed:
 1. A nanoparticle comprising a fusion protein thatcomprises a monomeric subunit protein joined to a Group 2 influenzavirus hemagglutinin (HA) protein, wherein at least 95% of the amino acidsequence of a head region of the HA protein is replaced with a linkersequence; wherein a helix A in a stem region of the HA protein isextended in length by the addition of helix-forming amino acid residues,thereby improving the stability of the fusion protein; and wherein thefusion protein comprises an amino acid sequence at least 80% identicalto SEQ ID NO:
 49. 2. The nanoparticle of claim 1, wherein an inter-helixloop in the stem region of the HA protein is replaced with a linkersequence.
 3. The nanoparticle of claim 1, wherein the stem region of theHA protein comprises one or more mutations that forms, or strengthens,an ionic interaction or a salt bridge within the HA protein.
 4. Thenanoparticle of claim 1, wherein the stem region of the HA proteincomprises one or more mutations that increases hydrophobic packingwithin the HA protein.
 5. The nanoparticle of claim 1, wherein themonomeric subunit protein is a ferritin monomeric subunit protein or alumazine synthase monomeric subunit protein.
 6. The nanoparticle ofclaim 1, wherein the fusion protein comprises an amino acid sequence setforth as SEQ ID NO:
 49. 7. The nanoparticle of claim 1, wherein thehelix A in the stem region of the HA protein is extended in length bythe addition of five helix-forming amino acid residues.
 8. A fusionprotein comprising a monomeric subunit protein joined to a Group 2influenza virus hemagglutinin (HA) protein, wherein at least 95% of theamino acid sequence of a head region of the HA protein is replaced witha linker sequence; wherein a helix A in the stem region of the HAprotein is extended in length by the addition of helix-forming aminoacid residues, thereby improving the stability of the fusion protein;and wherein the fusion protein comprises an amino acid sequence at least80% identical to SEQ ID NO:49.
 9. The fusion protein of claim 8, whereinan inter-helix loop in the stem region of the HA protein is replacedwith a linker sequence.
 10. The fusion protein of claim 8, wherein thestem region of the HA protein comprises one or more mutations thatforms, or strengthens, an ionic interaction or a salt bridge within theHA protein.
 11. The fusion protein of claim 8, wherein the stem regionof the HA protein comprises one or more mutations that increaseshydrophobic packing within the HA protein.
 12. The fusion protein ofclaim 8, wherein the monomeric subunit protein is a ferritin monomericsubunit protein or a lumazine synthase monomeric subunit protein.
 13. Anucleic acid molecule encoding the fusion protein of claim
 8. 14. Thefusion protein of claim 8, wherein the fusion protein comprises an aminoacid sequence set forth as SEQ ID NO:
 49. 15. A method of vaccinating anindividual against influenza virus, comprising administering aprophylactically or therapeutically effective amount of the nanoparticleof claim 1 to the individual.